Protein matrix materials, devices and methods of making and using thereof

ABSTRACT

The present invention relates to protein matrix materials and devices and the methods of making and using protein matrix materials and devices. More specifically the present invention relates to protein matrix materials and devices that may be utilized for various medical applications including, but not limited to, drug delivery devices for the controlled release of pharmacologically active agents, encapsulated or coated stent devices, vessels, tubular grafts, vascular grafts, wound healing devices including protein matrix suture material and meshes, skin/bone/tissue grafts, biocompatible electricity conducting matrices, clear protein matrices, protein matrix adhesion prevention barriers, cell scaffolding and other biocompatible protein matrix devices. Furthermore, the present invention relates to protein matrix materials and devices made by forming a film comprising one or more biodegradable protein materials, one or more biocompatible solvents and optionally one or more pharmacologically active agents. The film is then partially dried, rolled or otherwise shaped, and then compressed to form the desired protein matrix device.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a continuation of U.S. application Ser. No.09/796,170 filed on Feb. 28, 2001, which is a continuation in part ofU.S. application Ser. No. 09/160,421 filed on Sep. 25, 1998. This patentincorporates by reference the entire contents of the previouslymentioned applications and furthermore claims priority to andincorporates by reference herein the entire contents of U.S. ProvisionalApplication Ser. No. 60/185,420, filed Feb. 28, 2000, and U.S.Provisional Application Ser. No. 60/222,762, filed Aug. 3, 2000.

GOVERNMENTAL RIGHTS

At least a portion of the research described in this application wassupported in part by Governmental funding in the form of NIH Grant No.5R01GM51917. The Government has certain rights in the invention.

FIELD OF THE INVENTION

The present invention relates to protein matrix materials and devicesand the methods of making and using protein matrix materials anddevices. More specifically the present invention relates to proteinmatrix materials and devices that may be utilized for various medicalapplications including, but not limited to, drug delivery devices forthe controlled release of pharmacologically active agents, encapsulatedor coated stent devices, vessels, tubular grafts, vascular grafts, woundhealing devices including protein matrix suture material and meshes,skin/bone/tissue grafts, clear protein matrices, protein matrix adhesionprevention barriers, cell scaffolding and other biocompatible proteinmatrix devices. Furthermore, the present invention relates to proteinmatrix materials and devices made by forming a film comprising one ormore biodegradable protein materials, one or more biocompatible solventsand optionally one or more pharmacologically active agents. The film isthen partially dried, rolled or otherwise shaped, and then compressed toform the desired protein matrix device.

BACKGROUND OF THE INVENTION

Protein materials are generally present in the tissues of manybiological species. Therefore, the development of medical devices thatutilize protein materials, which mimic and/or are biocompatible with thehost tissue, have been pursued as desirable devices due to theiracceptance and incorporation into such tissue. For example theutilization of protein materials to prepare drug delivery devices,tissue grafts, wound healing and other types of medical devices havebeen perceived as being valuable products due to their biocompatibility.

The use of dried protein, gelatins and/or hydrogels have previously beenused as components for the preparation of devices for drug delivery,wound healing, tissue repair, medical device coating and the like.However, many of these previously developed devices do not offersufficient strength, stability and support when administered to tissueenvironments that contain high solvent content, such as the tissueenvironment of the human body. Furthermore, the features of such medicaldevices that additionally incorporated pharmacologically active agentsoften provided an ineffective and uncontrollable release of such agents,thereby not providing an optimal device for controlled drug delivery.

A concern and disadvantage of such devices is the rapid dissolving ordegradation of the device upon entry into an aqueous or high solventenvironment. For example, gelatins and compressed dry proteins tend torapidly disintegrate and/or lose their form when placed in an aqueousenvironment. Therefore, many dried or gelatin type devices do notprovide optimal drug delivery and/or structural and durabilitycharacteristics. Also, gelatins often contain large amounts of water orother liquid that makes the structure fragile, non-rigid and unstable.Alternatively, dried protein devices are often very rigid, tend to bebrittle and are extremely susceptible to disintegration upon contactwith solvents. It is also noted that the proteins of gelatins usuallydenature during preparation caused by heating, thereby reducing oreliminating the beneficial characteristics of the protein. Thedeficiencies gelatins and dried matrices have with regards to rapiddegradation and structure make such devices less than optimal for thecontrolled release of pharmacologically active agents, or for operatingas the structural scaffolding for devices such as vessels, stents orwound healing implants.

Hydrogel-forming polymeric materials, in particular, have been found tobe useful in the formulation of medical devices, such as drug deliverydevices. See, e.g., Lee, J. Controlled Release, 2, 277 (1985).Hydrogel-forming polymers are polymers that are capable of absorbing asubstantial amount of water to form elastic or inelastic gels. Manynon-toxic hydrogel-forming polymers are known and are easy to formulate.Furthermore, medical devices incorporating hydrogel-forming polymersoffer the flexibility of being capable of being implantable in liquid orgelled form. Once implanted, the hydrogel forming polymer absorbs waterand swells. The release of a pharmacologically active agent incorporatedinto the device takes place through this gelled matrix via a diffusionmechanism.

However, many hydrogels, although biocompatible, are not biodegradableor are not capable of being remodeled and incorporated into the hosttissue. Furthermore, most medical devices comprising of hydrogelsrequire the use of undesirable organic solvents for their manufacture.Residual amounts of such solvents could potentially remain in themedical device, where they could cause solvent-induced toxicity insurrounding tissues or cause structural or pharmacological degradationto the pharmacologically active agents incorporated within the medicaldevice. Finally, implanted medical devices that incorporatepharmacologically active agents in general, and such implanted medicaldevices comprising hydrogel-forming polymers in particular, oftentimesprovide suboptimal release characteristics of the drug(s) incorporatedtherein. That is, typically, the release of pharmacologically activeagents from an implanted medical device that includes pharmacologicallyactive agent(s) is irregular, e.g., there is an initial burst periodwhen the drug is released primarily from the surface of the device,followed by a second period during which little or no drug is released,and a third period during which most of the remainder of the drug isreleased or alternatively, the drug is released in one large burst.

It would be desirable to provide a medical device that wouldbiocompatibly degrade and resorb into the host tissue for which it isadministered. Alternatively, it would be desirable to provide a medicaldevice that can be incorporated and remodeled by the host tissue toremain in the tissue and provide a prolonged intended function of thedevice. Furthermore, it would be desirable to provide improved medicaldevices capable of sustained, controlled local delivery ofpharmacologically active agents when implanted while also beingbiodegradable and resorbable or alternatively capable of beingincorporated and remodeled into the host tissue, such that removal ofthe device is not necessary. It would further be desirable to controlthe rate of delivery from such devices to avoid possible side effectsassociated with irregular delivery, e.g., high drug concentrationinduced tissue toxicity. Finally, it would be advantageous if suchdevices could be manufactured with biocompatible proteins and solventsso that the potential for residual solvent toxicity and immunogenicityis reduced.

SUMMARY OF THE INVENTION

The present invention relates to protein matrix materials and devicesand the methods of making and using protein matrix materials anddevices. Embodiments of the present invention may include, but are notlimited to, drug delivery devices for the controlled release ofpharmacologically active agents, encapsulated or coated stent devices,vessels, tubular grafts, vascular grafts, wound healing devicesincluding protein matrix suture material and meshes, skin/bone/tissuegrafts, clear protein matrices, protein matrix adhesion preventionbarriers, cell scaffolding and other biocompatible protein matrixdevices.

Furthermore, the present invention relates to a method of making aprotein matrix material and devices by forming a coatable compositioncomprising one or more biocompatible protein materials, one or morebiocompatible solvents and optionally one or more pharmacologicallyactive agents. The coatable composition may also include additionalpolymeric materials and/or therapeutic entities that would provideadditional beneficial characteristics or features to the protein matrix.The coatable composition is then coated so as to form a film (preferablya substantially planar body having opposed major surfaces and preferablyhaving a thickness between the major surfaces of from about 0.1millimeters to about 5 millimeters). Next, the film is at leastpartially dried until it is cohesive, and then formed (rolled, folded,accordion-pleated, crumpled, or otherwise shaped) into a cohesive bodyhaving a surface area less than that of the film. The cohesive body isthen compressed to provide the desired protein matrix device inaccordance with the present invention.

The protein matrix material is compressed to limit bulk biocompatiblesolvent, such as bulk or trapped water (i.e., iceberg water). Theelimination of the bulk biocompatible solvent by compressing enhancesthe strength and durability of the matrix by initiating, stimulating andforcing additional intramolecular and intermolecular attraction betweenthe biocompatible solvent molecules, such as hydrogen bonding activity,and also initiates, stimulates and forces intramolecular andintermolecular activity between the protein molecules, the biocompatiblesolvent molecules and the optional pharmacologically active agents.

The above described process has many advantages if one or morepharmacologically active agents are incorporated into the matrix. Forexample, the controlled release characteristics of the protein matrixprovides for a higher amount of pharmacologically active agent(s) thatmay be incorporated into the matrix. Additionally, the pharmacologicallyactive agent(s) is/are substantially homogeneously distributedthroughout the protein matrix material or device. This homogenousdistribution provides for a more systematic and consistent release ofthe pharmacologically active agent(s). As a result, the releasecharacteristics of the pharmacologically active agent from the proteinmatrix material and/or device are enhanced.

As previously suggested, embodiments of the protein matrix devicesproduced utilizing the method of the present invention are capable ofthe sustainable, controllable local delivery of pharmacologically activeagent(s), while also providing the advantage of being capable of beingdegraded, and preferably safely resorbed. The resorbable characteristicof various embodiments of the present invention eliminates the need forthe removal of the drug delivery device from the patient once thepharmacologically active agent(s) have been completely delivered fromthe matrix.

Additionally, other embodiments of the present invention may be producedto remain in the patient. This may be accomplished by utilizing proteinmaterials that do not readily degrade and resorb, but are remodeled bythe host tissue, by incorporating an additional polymeric material intothe protein matrix or by treating the protein matrix material with areagent. For example, the protein matrix material may be partially ortotally treated with a reagent, such as glutaraldehyde, to createcrosslinking of the protein fibers in the matrix. The crosslinking ofthe protein material may be utilized to produce a biocompatible devicethat has a desired function, form or shape, such as a graft, valve ortube, and additionally may retain its form without resorbing ordegrading into the patient or until the matrix has been incorporatedand/or remodeled into the host tissue. Examples of protein matrixdevices that would benefit from such a nonresorbable or nondegradablecharacteristic include, but are not limited to, stent covers, vessels,valves, tissue grafts, electronic implant coverings and other devicesthat need a biocompatible sustaining structure to remain in the patient.Such devices may further include one or more pharmacologically agents.The nonresorbable and nondegradable protein matrix device would stillretain the systematic release of the pharmacological active agents,thereby diffusing out of the device rather than releasing upondegradation of the protein matrix material.

Whether the device is intended to be entirely resorbable or not, themethod of making the protein matrix devices is generally the same. Indescribing the method more specifically, the method comprises the stepsof preparing a coatable composition comprising one or more biodegradableprotein materials, one or more biocompatible solvents and optionally oneor more pharmacologically active agents. Additional biodegradablepolymeric materials may be added in the preparation of the coatablecomposition to provide optimum features desired for the particularprotein matrix device being prepared. For example, polyanhydride may beadded to the protein matrix to inhibit the absorption of physiologicalbody fluids and slows the diffusion and/or degradation of the proteinmatrix and/or pharmacological active agent. Preferably, thebiocompatible solvent is water, dimethyl sulfoxide (DMSO), ethanol, anoil, combinations of these, or the like. More preferably, thebiocompatible solvent comprises water. The coatable composition is thencoated to form a film and partially dried until the coated film can beformed into a cohesive body, e.g., preferably until the film has asolvent content of from about 50% to about 70%. The film is then formedinto the cohesive body, preferably with a surface area less than that ofthe film. The film is then shaped into a cohesive body, e.g., rolled,folded, accordion-pleated, crumpled, or otherwise shaped into a cylinderor shaped into a ball, cube and the like, preferably with a surface arealess than that of the film. The cohesive body is then compressed toremove as much of the solvent as possible so that the compressed bodyremains cohesive, but without removing so much solvent that thecompressed body becomes brittle or otherwise lacks cohesiveness.Typically, the resulting protein matrix device has a solvent content offrom about 10% to about 60%, preferably from about 30% to about 50%. Ifdesired, the compressed body may next be treated with a crosslinkingreagent, such as glutaraldehyde to form a compressed body that hasadditional structural and nonresorbable features.

As previously suggested, by coating the aforementioned components into afilm, partially drying the film, forming the film into a cohesive bodyand subsequently compressing the cohesive body, a protein matrix device,which includes one or more pharmacologically active agents, has asubstantially homogeneous distribution of the pharmacologically activeagent(s). Due to this substantially homogeneous distribution, theprotein matrix devices of the present invention that include one or morepharmacologically active agents provide a sustainable and controllablerelease of the pharmacologically active agent(s). Furthermore, themethod of the present invention utilizes biocompatible, and if selected,resorbable and biodegradable, protein materials. As a result, proteinmatrix devices formed in accordance with the method of the presentinvention may include the benefit of remaining in the patientindefinitely or simply resorbing and/or degrading into the tissuesurrounding it. Finally, since the protein matrix material isbiocompatible, any solvent remaining in the protein matrix device afterthe manufacture thereof presents a reduced, if not substantiallyeliminated, risk of producing undesirable side effects when implantedinto a patient.

The biocompatible protein material incorporated into a device inaccordance with the present invention generally comprises one or morebiocompatible proteins, which preferably are a water-absorbing,biocompatible protein. Additionally, the biocompatible protein may besynthetic, genetically engineered or natural. In various embodiments ofthe present invention, the genetically engineered protein materialcomprises silklike blocks and elastinlike blocks. As previouslyindicated, the protein matrix device can incorporate any desiredpharmacologically active agent or even a second drug delivery device,e.g., corticosteroids, opioid analgesics, neurotoxins, localanesthetics, vesicles, lipospheres, microspheres, nanospheres, enzymes,combinations of these, and the like.

It has now additionally been discovered that the sustainable release andrate controllable characteristics of the present protein matrix devicemay also been beneficially utilized to deliver other drug deliverydevices that are either vulnerable to migration from the delivery siteand/or are potentially undesirably reactive with surrounding bodilyfluids or tissues. That is, not only can the protein matrix device ofthe present invention be beneficially utilized to deliver apharmacologically active agent to a particular site where a therapeuticeffect is desired, but also the protein matrix device of the presentinvention may be a “two-stage drug delivery device” utilized to delivera second, migration-vulnerable drug delivery device comprising apharmacologically active agent so that the second, migration-vulnerableand/or reactive drug delivery device is held in place, e.g., by theprotein matrix provided by the protein matrix device of the presentinvention. In the instance that the two-stage protein matrix device isused to deliver a reactive drug delivery device, the protein matrix ofthe two stage drug delivery device reduces, if not substantiallyprevents the second drug delivery device from undesirably reacting withsurrounding bodily tissues and/or fluids.

Thus, in another aspect, the present invention provides a protein matrixdevice comprising a compressed matrix comprising at least onebiodegradable polymeric material and at least one such substancevulnerable to migration and/or reaction with surrounding tissues orbodily fluids, wherein said substance is substantially homogeneouslydistributed within the matrix. Examples of such substances include, butare not limited to, vesicles, such as lipospheres or liposomes,comprising an encapsulated pharmacologically active agent, microspherescomprising an encapsulated pharmacologically active agent, combinationsof these, and the like. Other examples of such substances include, butare not limited to, stents, electronic devices and other non-tissueimplant that may illicit an adverse reaction from surrounding tissues.

Inasmuch as the protein matrix devices of the present invention providethe sustained release of one or more pharmacologically active agents ina rate controllable fashion, they are also capable of delivering othermigration-vulnerable and/or reactive drug delivery devices andfurthermore are produced in a manner that reduces, if not eliminates,the risk of residual solvent toxicity or adverse tissue reaction. Also,the protein matrix devices of the present invention provide a method ofeffecting a local therapeutic response in a patient in need of suchtreatment. Specifically, the method comprises the step of administeringa protein matrix device in accordance with the present invention to thesite at which a local therapeutic response is desired. Additionally, theprotein matrix devices may be administered for systemic delivery ofpharmacologically active agents, including oral, as well as nasal,pulmonary, subcutaneous, or any other parenteral mode of delivery.Preferably, the therapeutic response effected is an analgesic response,an anti-inflammatory response, an anesthetic response, a responsepreventative of an immunogenic response, an anti-coagulatory response, agenetic response, a protein assembly response, an antibacterialresponse, a vaccination response, combinations of these, and the like.As used herein, unless stated otherwise, all percentages are percentagesbased upon the total mass of the composition being described, e.g., 100%is total.

BRIEF DESCRIPTION OF THE FIGURES

The above mentioned and other advantages of the present invention, andthe manner of attaining them, will become more apparent and theinvention itself will be better understood by reference to the followingdescription of the embodiments of the invention taken in conjunctionwith the accompanying drawing, wherein:

FIG. 1 is a schematic illustration, in partial cross-sectional view, ofa compression molding device that may be used in the method of thepresent invention in a configuration prior to compression;

FIG. 2 is a schematic illustration, in partial cross-sectional view, ofa compression molding device that may be used in the method of thepresent invention in a configuration during compression;

FIG. 3 is a schematic illustration, in partial cross-sectional view, ofa compression molding device that may be used in the method of thepresent invention in a configuration during ejection; and

FIG. 4 depicts an embodiment of a drug delivery device of the presentinvention in particulate form;

FIG. 5 depicts an embodiment of a drug delivery device of the presentinvention in particulate form;

FIG. 6 is a schematic illustration, in partial cross-sectional view, ofa compression molding device that may be used in the method of thepresent invention in wherein the inner insert includes a mandrel thatthat is engaged with a stent.

FIG. 7 depicts various views of an embodiment of the present inventionformulated as a tubular graft;

FIG. 8 depicts various embodiments of an encapsulated stent device witha silastic tube and/or angioplasty balloon inserted therein;

FIG. 9 depicts various embodiments of an encapsulated stent device;

FIG. 10 depicts various views of a multi-layer vessel;

FIG. 11 depicts an embodiment of a tubular graft that illustrates thecapability, compliancy and capacity of the protein matrix material toaccept sutures and reform to its original shape;

FIG. 12 depicts an embodiment of a compression molding device whereinthe inner insert includes a mandrel;

FIG. 13 depicts the top view of an embodiment of the compression moldingdevice without the upper insert or plunger;

FIG. 14 depicts an embodiment of a wound healing device shaped in theconfiguration of an ultra-thin skin graft matrix;

FIG. 15 depicts an embodiment of a wound healing device comprising aprotein matrix that is positioned in the center of a non-adhesive stripof material attached to two adhesive ends;

FIG. 16 depicts an embodiment of a protrusion device 34 that includes aport seal;

FIG. 17 is a graphical illustration of the in vitro releasecharacteristics of the pharmacologically active agent, sufentanil, froma drug delivery device in accordance with the present invention.

FIG. 18 is a before and after depiction of an embodiment of a proteinmatrix device that includes a release mechanism;

FIG. 19 depicts two protein matrix devices that include releasemechanisms contained in an agar gel;

FIG. 20 depicts a time progression illustration of a protein matrixdevice that includes a protein matrix device following release of themechanism;

FIG. 21 is a magnified view of an embodiment of a noncrosslinked wafer;

FIG. 22 is a magnified view of an embodiment of a crosslinked wafer;

FIG. 23 is a chart of the effect of GA crosslinking and molding pressureon the Young's modulus of collagen wafers;

FIG. 24 is a chart of the effect of GA crosslinking and molding pressureon the UTS of collagen wafers;

FIG. 25 is a chart regarding the amount of collagen released into PBSinvolving noncrosslinked embodiments of the present invention;

FIG. 26 is a chart regarding the amount of collagen released into PBSinvolving various embodiments of the present invention crosslinked bycontacting with 1% glutaraldehyde for 1 minute;

FIG. 27 is a chart regarding the amount of collagen released into PBSinvolving various embodiments of the present invention crosslinked bycontacting with 1% glutaraldehyde for 10 minutes;

FIG. 28 is a chart regarding the amount of collagen released into PBSinvolving various embodiments of the present invention crosslinked bycontacting with 1% glutaraldehyde for 30 minutes;

FIG. 29 depicts an embodiment of a vascular tube;

FIG. 30 depicts an embodiment of a vascular tube tested for durabilityand compliance;

FIG. 31 depicts views of both sides of an embodiment of a vascular tubetested for hydraulic pressure;

FIG. 32 depicts, at the arrows, an embodiment of a bulging vascular tubetested for pressure strength and durability; and

FIG. 33 depicts the results of the hEGF release study from embodimentsof PVA particles used in the protein matrix wafers of the presentinvention.

DETAILED DESCRIPTION OF THE INVENTION

The embodiments of the invention described below are not intended to beexhaustive or to limit the invention to the precise forms disclosed inthe following detailed description. Rather, the embodiments are chosenand described so that others skilled in the art may appreciate andunderstand the principles and practices of the present invention. Thepresent invention relates to protein matrix materials and devices and amethod of making such protein matrix materials and devices. Morespecifically, the method of the present invention involves preparing acoatable composition comprising one or more biocompatible proteinmaterials, one or more biocompatible solvents and optionally one or morepharmacologically active agents. It is noted that additional polymericmaterials and/or therapeutic entities may be included in the coatablecomposition to provide various beneficial features such as strength,elasticity, structure and/or any other desirable characteristics. Thecoatable composition is then coated to form a film that is subsequentlypartially dried, formed into a cohesive body, and then compressed toprovide a protein matrix device in accordance with the presentinvention.

While not wishing to be bound by any theory, it is believed that bypreparing a coatable composition from the aforementioned components,coating this composition to form a film that is subsequently partiallydried, and then forming the film into a cohesive body, a relativelyhomogeneous distribution of the components is obtained in the cohesivebody. Furthermore, when the film has dried enough so as to be cohesiveunto itself, e.g., to a solvent content from about 50% to about 70%,subsequently formed into a cohesive body and then compressed many, ifnot all, of any distribution anomalies are removed or resolved.Therefore, when the protein matrix device includes a pharmacologicallyactive agent, the distribution of the pharmacologically active agent isrendered substantially homogenous throughout the resulting drug deliverydevice.

In addition, the removal of such distribution anomalies also includesthe removal of bulk or trapped biocompatible solvent, such as aqueoussolutions, i.e. bulk water (i.e., iceberg water) from the matrix. Forexample, in aqueous solutions, proteins bind some of the water moleculesvery firmly and others are either very loosely bound or form islands ofwater molecules between loops of folded peptide chains. Because thewater molecules in such an island are thought to be oriented as in ice,which is crystalline water, the islands of water in proteins are calledicebergs. Furthermore, water molecules may also form bridges between thecarbonyl (C═O) and imino (NH) groups of adjacent peptide chains,resulting in structures similar to those of a pleated sheet (β-sheets)but with a water molecule in the position of the hydrogen bonds of thatconfiguration. Generally, the amount of water bound to one gram of aglobular protein in solution varies from 0.2 to 0.5 grams. Much largeramounts of water are mechanically immobilized between the elongatedpeptide chains of fibrous proteins, such as gelatin. For example, onegram of gelatin can immobilize at room temperature 25 to 30 grams ofwater. It is noted that other biocompatible solvents may also interactwith protein molecules to effect intra- and inter-molecular forces uponcompression. The compression of the cohesive body removes the bulksolvent from the resulting protein matrix.

The protein matrix of the present invention traps biocompatible solventmolecules, such as water molecules, and forces them to interact with theprotein to produce a protein-water matrix with natural physical,biological and chemical characteristics. The compression of the cohesivebody eliminates the islands of water or bulk water resulting in astrengthened protein matrix structure. Furthermore, the elimination ofbulk water enhances the homogenous characteristics of the protein matrixby reducing the pooling of water and spacing of the protein moleculesand pharmacologically active agent molecules. Upon compression of thecohesive body, the remaining water molecules are forced to interact withmost to all protein molecules and thereby add strength, structure andstability to the protein matrix. The compression forces out most of thenon-structural bulk water (immobilized water) from the matrix. Aspreviously suggested, the bulk water is extra water that is only looselybound to the matrix. The water that interacts with the protein moleculesof the protein matrix reduces and/or prevents the protein fromdenaturing during compression and facilitates the protein binding withthe water through intra- and inter-molecular forces (i.e., ionic,dipole-dipole such as hydrogen bonding, London dispersion, hydrophobic,etc.). The enhanced binding characteristics of the protein matrixfurther inhibits the loss of non-bulk solvent molecules that interactwith protein molecules. Experiments have indicated that a protein matrixdries to 25-45% water during overnight drying processes that wouldnormally dry over 100 times that same amount of water if it were not inthe matrix.

Furthermore, the resulting protein matrix device preferably has aslittle solvent as possible while still being cohesive and possessing thedesired features relevant to the device's function, e.g., preferably asolvent content of from about 10% to about 60%, more preferably asolvent content of from about 30% to about 50%. It is found that when aprotein matrix device of the present invention includes apharmacologically active agent, the partial drying of the film to form acohesive body and subsequent compressing of the cohesive body, forcesmore solvent out of the body, thereby producing a resulting proteinmatrix device that has a significantly higher concentration ofpharmacologically active agent relative to other components of thedevice than is obtainable in protein matrix devices produced by othermethods. As a result of the substantially uniform dispersion of agreater concentration of pharmacologically active agent, a sustained,controlled release of the pharmacologically active agent is achieved,while reducing the initial high concentration effects that can beassociated with other devices that include pharmacologically activeagents or bolus injections of pharmacologically active agents.

Reducing the solvent content has the additional effect that theresulting drug delivery device is more structurally sound, easy tohandle, and thus, easy to insert or implant. Upon insertion, the cellsof the tissue contacting the implanted protein matrix holds the proteinmatrix device substantially in the desired location. Alternatively,embodiments of the protein matrix may be held in the desired location bytissue contact, pressure, sutures, adhesives and/or tissue folds orcreases. Embodiments of the protein matrix device may biodegrade andresorbs over time or retain their structural integrity.

To form the coatable composition, the biocompatible protein material(s),the biocompatible solvent(s), and optionally the pharmacologicallyactive agent(s) may be combined in any manner. It is noted that one ormore additional polymeric materials and/or therapeutic entities may beadded to the coatable composition during the combination step to provideadditional desirable characteristics to the coatable composition. Forexample, the components may simply be combined in one step, oralternatively, the biocompatible protein materials may be dissolvedand/or suspended in a biocompatible solvent and an additional proteinmaterial and/or the pharmacologically active agent may be dissolvedand/or suspended in the same or another biocompatible solvent and thenthe resulting two solutions mixed.

Once prepared, the coatable composition may be coated onto any suitablesurface from which it may be released after drying by any suitablemethod. Examples of suitable coating techniques include spin coating,gravure coating, flow coating, spray coating, coating with a brush orroller, screen printing, knife coating, curtain coating, slide curtaincoating, extrusion, squeegee coating, and the like. The coated film(preferably having a substantially planar body having opposed majorsurfaces) is desirably thin enough so as to be capable of drying withina reasonable amount of time and also thin enough so that the film can beformed into a cohesive body comprising a substantially homogeneousdispersion of the components of the coatable composition. For example, athinner film will tend to form a more homogeneous cohesive body when thefilm is formed into the shape of a cylinder. A typical coated film ofthe coatable composition have a thickness in the range of from about0.01 millimeters to about 5 millimeters, more preferably from about 0.05millimeters to about 2 millimeters.

Initially, when the film is first coated, it is likely to benon-cohesive, fluidly-flowable, and/or non self-supporting. Thus, thecoated film is preferably dried sufficiently so that it becomescohesive, i.e., the film preferably sticks to itself rather than othermaterials. The film may simply be allowed to dry at room temperature, oralternatively, may be dried under vacuum, conditions of mild heating,i.e., heating to a temperature of from about 25° C. to about 50° C., orconditions of mild cooling, i.e. cooling to a temperature of from about0° C. to about 10° C. When utilizing heat to dry the film, care shouldbe taken to avoid denaturation or structural degradation of thepharmacologically active agent incorporated therein.

The specific solvent content at which the film becomes cohesive untoitself will depend on the individual components incorporated into thecoatable composition. Generally, films that have too high of a solventcontent will not be cohesive. Films that have too low of a solventcontent will tend to crack, shatter, or otherwise break apart uponefforts to form them into a cohesive body. With these considerations inmind, the solvent content of a partially dried film will preferably befrom about 20% to about 80%, more preferably from about 30% to about 65%and most preferably from about 35% to about 50%.

Once the film is capable of forming a cohesive body, such a cohesivebody may be formed by any of a number of methods. For example, the filmmay be rolled, folded, accordion-pleated, crumpled, or otherwise shapedsuch that the resulting cohesive body has a surface area that is lessthan that of the coated film. For example the film can be shaped into acylinder, a cube, a sphere or the like. Preferably, the cohesive body isformed by rolling the coated film to form a cylinder.

Once so formed, the cohesive body is compressed to form a protein matrixdevice in accordance with the present invention. Any manually orautomatically operable mechanical, pneumatic, hydraulic, or electricalmolding device capable of subjecting the cohesive body to pressure issuitable for use in the method of the present invention. In theproduction of various embodiments of the present invention, a moldingdevice may be utilized that is capable of applying a pressure of fromabout 100 pounds per square inch (psi) to about 100,000 psi for a timeperiod of from about 2 seconds to about 48 hours. Preferably, themolding device used in the method of the present invention will becapable of applying a pressure of from about 1000 psi to about 30,000psi for a time period of from about 10 seconds to about 60 minutes. Morepreferably, the molding device used in the method of the presentinvention will be capable of applying a pressure of from about 3,000 psito about 25,000 psi for a time period of from about one minute to aboutten minutes.

Compression molding devices suitable for use in the practice of themethod of the present invention are generally known. Suitable devicesmay be manufactured by a number of vendors according to providedspecifications, such as desirable pressure, desired materials forformulation, desired pressure source, desired size of the moldable andresulting molded device, and the like. For example, Gami Engineering,located in Mississauga, Ontario manufactures compression molding devicesto specifications provided by the customer. Additionally, manycompression molding devices are commercially available.

An embodiment of a compression molding device 10 suitable for use in themethod of the present invention is schematically shown in FIG. 1.Compression molding device 10 is equipped with a mold body 12 in whichcohesive body 22 can be subjected to pressure in order to compress andmold the cohesive body 22 into a protein matrix device in accordancewith the present invention. Mold body 12 is shown supported in positionon a base plate 20. More specifically, mold body 12 has provided thereina cavity 16 that preferably extends all the way through mold body 12.Within the cavity 16 a molding chamber 17 can be defined into which acohesive body in accordance with the present invention may be inserted.The molding chamber 17 may be configured in any shape and size dependingupon the shape and size of the protein matrix device. For example, thechamber may take the shape or form of a tube, heart valve, cylinder orany other desired shape. The cavity 16 may comprise a bore of any shapethat may be machined, formed, cast or otherwise provided into the moldbody 12. The compression molding device may optionally include one ormore apertures of approximately 0.004 to 0.0001 inches for biocompatiblesolvent to escape the chamber 17 during compression of the cohesivebody. An inner insert 18 is preferably slidably fit within cavity 16 tobe positioned against one surface 13 of the base plate 20 to define themolding chamber 17 and support to cohesive body 22 when positionedwithin the molding chamber 17. The insert 18 may be any shape that isdesired for molding the protein matrix device. For example the insert 18may be a solid cylindrical mandrel that can form the lumen of a tube orvessel. The insert 18 is thus fixed with respect to the mold body 12 todefine the inner extent of the molding chamber 17. An outer insert 19 isalso preferably provided to be slidable within the cavity 16.

Outer insert 19 is used to close the molding chamber 17 of cavity 16after the inner insert 18 and the cohesive body 22 are provided in thatorder within the cavity 16. The inner and outer inserts 18 and 19,respectively, can be the same or different from one another, but bothare preferably slidably movable within the cavity 16. The inner andouter inserts 18 and 19, respectively, are configured to create thedesired form or shape of the protein matrix device. Additionally, theinserts 18 and 19 may be shaped similarly to the shape of the cavity 16to slide therein and are sized to effectively prevent the material ofthe cohesive body 22 to pass between the inserts 18 and 19 and the wallsof cavity 16 when the cohesive body 22 is compressed as described below.However, the sizing may be such that moisture can escape between theouter edges of one or both inserts 18 and 19 and the surface walls ofthe cavity 16 from the cohesive body 22 during compression. Otherwise,other conventional or developed means can be provided to permit moistureto escape from the mold cavity during compression. For example, smallopenings could pass through one or both of the inserts 18 and 19 or moldbody 12 which may also include one-way valve devices. Insert 18 may beeliminated so that surface 13 of base plate 20 defines the lowerconstraint to molding chamber 17. However, the use of insert 18 isbeneficial, in that its presence facilitates easy removal of thecohesive body 22 after compression (described below) and provides asufficiently hard surface against which the cohesive body 22 can becompressed. Moreover, by utilizing a series of differently sized and/orshaped inner inserts 18, the volume of the molding chamber can bevaried, or different end features may be provided to the cohesive body22. Outer inserts 19 can likewise be varied.

Outer insert 19 is also positioned to be advanced within cavity 16 orretracted from cavity 16 by a plunger 14. Preferably, the contactingsurfaces of outer insert 19 and plunger 14 provide a cooperatingalignment structure so that pressure can be evenly applied to thecohesive body 22. The plunger 14 may comprise a part of, or may beoperatively connected with a pressure generation mechanism 24 that hasthe ability to apply pressure of the type and force necessary to achievethe results of the present invention. Conventional or developedtechnologies are contemplated, such as using mechanical, hydraulic,pneumatic, electrical, or other systems. Such systems can be manually orautomatically operable.

Plunger 14 operates independently of mold body 12 and is operationallycoupled to the pressure generation mechanism 24. Pressure generationmechanism 24 may be any pressure source capable of applying from about100 psi to about 100,000 psi for a time period of from about 2 secondsto about 48 hours, preferably capable of applying from about 1000 psi toabout 30,000 psi for a time period of from about 10 seconds to about 60minutes, and more preferably, capable of applying a pressure of fromabout 3000 psi to about 25,000 psi for a time period of from about 1minute to about 10 minutes. Preferably, plunger 14 is formulated of amaterial capable of translating substantially all of the pressureapplied by pressure generation mechanism 24 to cohesive body 22.

Mold body 12 may be fabricated from any material capable of withstandingthe pressure to be applied from pressure generation mechanism 24, e.g.,high density polyethylene, Teflon®, steel, stainless steel, titanium,brass, copper, combinations of these and the like. Desirably, mold body12 is fabricated from a material that provides low surface friction toinserts 18 and 19 and cohesive body 22. Alternatively, surfaces definingthe cavity 16 may be coated with a low friction material, e.g., Teflon®,to provide such low surface friction. Due to its relatively low cost,sufficient strength and surface friction characteristics, mold body 12is desirably fabricated from brass. Cavity 16, extending substantiallythrough mold body 12, may be of any shape and configuration, asdetermined by the desired configuration of the resulting, compressedprotein matrix devices. In one embodiment, cavity 16 is cylindrical.However, the shape of the cavity 16 can be configured to accommodate theshape and size of the resulting, compressed protein matrix device. Asabove, inserts 18 and 19 preferably fit within cavity 16 in a mannerthat allows moisture to escape from mold body 12, and so that inserts 18and 19 may be easily inserted into and removed from cavity 16.Furthermore, it is preferred that inserts 18 and 19 fit within cavity 16in a manner that provides adequate support and containment for cohesivebody 22, so that, upon compression, the material of cohesive body 22does not escape cavity 16 in a manner that would produce irregularlyshaped edges on the resulting protein matrix device.

According to one procedure for using compression molding device 10 tocarry out the method of the present invention, the mold body 12 ispositioned as shown in FIG. 1 on the base plate 20, which itself may besupported in any manner. Then, an inner insert 18 is placed into cavity16 followed by a cohesive body 22 to be compressed and an outer insert19 as shown. Plunger 14 is then positioned so as to be in drivingengagement with outer insert 19. Then, as schematically illustrated inFIG. 2, the pressure generation mechanism 24 is activated to moveplunger 14 in the direction of arrow A to reduce the volume of themolding cavity 17 to make a compressed cohesive body 23. Pressuregeneration mechanism 24 applies sufficient pressure, i.e., from about100 psi to about 100,000 psi for a time period of from about 2 secondsto about 48 hours, to plunger 14, insert 19 and cohesive body 22 againstthe inner insert 18, thereby driving moisture from and compressingcohesive body 22 into a protein matrix device in accordance with thepresent invention.

As shown in FIG. 3, the compressed cohesive body 23 can then be ejectedfrom the mold body 12 along with inserts 18 and 19 by positioning themold body 12 on a support spacer 30 and further advancing the plunger 14in the direction of arrow A by the pressure generation mechanism 24.Generally, base plate 20 is separated from the mold body 12 whenejecting the protein matrix device and inserts 18 and 19. The supportspacer 30 is preferably shaped and dimensioned to provide an open volume31 for the compressed cohesive body 23 to be easily removed. That is,when the plunger 14 is sufficiently advanced, the insert 18 andcompressed cohesive body 23 can fall into the open volume 31 within thesupport spacer 30. After completion, the plunger 14 can be fullyretracted so that the compression molding device 10 can be reconfiguredfor a next operation.

Any biocompatible protein material may be utilized in the protein matrixdevices and corresponding methods of the present invention. Preferably,any such material will at least be water-compatible, and more preferablywill be water-absorbing or hydrogel forming. Furthermore, one or morebiocompatible protein materials may be incorporated into the proteinmatrix device of the present invention and may desirably be selectedbased upon their biocompatible and/or degradation properties. Thecombination of more than one biocompatible protein can be utilized tomimic the environment in which the device is to be administered,optimize the biofunctional characteristics, such as cell attachment andgrowth, nonimmuno-response reaction and/or alter the releasecharacteristics, or duration of an included pharmacologically activeagent, if a pharmacologically active agent is to be included in thedevice.

The biocompatible protein material comprises one or more biocompatiblesynthetic protein, genetically-engineered protein, natural protein orany combination thereof. In many embodiments of the present invention,the biocompatible protein material comprises a water-absorbing,biocompatible protein. In various embodiments of the present invention,the utilization of a water-absorbing biocompatible protein provides theadvantage that, not only will the protein matrix device bebiodegradable, but also resorbable. That is, that the metabolites of thedegradation of the water-absorbing biodegradable protein may be reusedby the patient's body rather than excreted. In other embodiments that donot degrade or resorb the water absorbing material provides enhancedbiocompatible characteristics since the device is generally administeredto environments that contain water.

The biocompatible protein utilized may either be naturally occurring,synthetic or genetically engineered. Naturally occurring protein thatmay be utilized in the protein matrix device of the present inventioninclude, but are not limited to elastin, collagen, albumin, keratin,fibronectin, silk, silk fibroin, actin, myosin, fibrinogen, thrombin,aprotinin, antithrombin III and any other biocompatible natural protein.It is noted that combinations of natural proteins may be utilized tooptimize desirable characteristics of the resulting protein matrix, suchas strength, degradability, resorption, etc. Inasmuch as heterogeneityin molecular weight, sequence and stereochemistry can influence thefunction of a protein in a protein matrix device, in some embodiments ofthe present invention synthetic or genetically engineered proteins arepreferred in that a higher degree of control can be exercised over theseparameters.

Synthetic proteins are generally prepared by chemical synthesisutilizing techniques known in the art. Examples of such syntheticproteins include but are not limited to natural protein madesynthetically and collagen linked GAGS like collagen-heparin,collagen-chondroitin and the like. Also, individual proteins may bechemically combined with one or more other proteins of the same ordifferent type to produce a dimer, trimer or other multimer. A simpleadvantage of having a larger protein molecule is that it will makeinterconnections with other protein molecules to create a strongermatrix that is less susceptible to dissolving in aqueous solutions.

Additional, protein molecules can also be chemically combined to anyother chemical so that the chemical does not release from the matrix. Inthis way, the chemical entity can provide surface modifications to thematrix or structural contributions to the matrix to produce specificcharacteristics. The surface modifications can enhance and/or facilitatecell attachment depending on the chemical substance or the cell type.The structural modifications can be used to facilitate or impededissolution, enzymatic degradation or dissolution of the matrix.

Synthetic biocompatible materials may be cross-linked, linked, bonded orchemically and/or physically linked to pharmacological active agents andutilized alone or in combination with other biocompatible proteins toform the cohesive body. Examples of such cohesive body materialsinclude, but are not limited to heparin-protein, heparin-polymer,chondroitin-protein, chondroitin-polymer, heparin-cellulose,heparin-alginate, heparin-polylactide, GAGs-collagen, heparin-collagen.

Specific examples of a particularly preferred genetically engineeredproteins for use in the protein matrix devices of the present inventionis that commercially available under the nomenclature “ELP”, “SLP”,“CLP”, “SLPL”, “SLPF” and “SELP” from Protein Polymer Technologies, Inc.San Diego, Calif. ELP's, SLP's, CLP's, SLPL's, SLPF's and SELP's arefamilies of genetically engineered protein polymers consisting ofsilklike blocks, elastinlike blocks, collagenlike blocks, lamininlikeblocks, fibronectinlike blocks and the combination of silklike andelastinlike blocks, respectively. The ELP's, SLP's, CLP's, SLPL's,SLPF's and SELP's are produced in various block lengths andcompositional ratios. Generally, blocks include groups of repeatingamino acids making up a peptide sequence that occurs in a protein.Genetically engineered proteins are qualitatively distinguished fromsequential polypeptides found in nature in that the length of theirblock repeats can be greater (up to several hundred amino acids versusless than ten for sequential polypeptides) and the sequence of theirblock repeats can be almost infinitely complex. Table A depicts examplesof genetically engineered blocks. Table A and a further description ofgenetically engineered blocks may be found in Franco A. Ferrari andJoseph Cappello, Biosynthesis of Protein Polymers, in: Protein-BasedMaterials, (eds., Kevin McGrath and David Kaplan), Chapter 2, pp. 37-60,Birkhauser, Boston (1997).

TABLE A Protein polymer sequences Polymer Name Monomer Amino AcidSequence SLP 3 [(GAGAGS)₉ GAAGY)] SLP 4 (GAGAGS)_(n) SLP F [(GAGAGS)₉GAA VTGRGDSPAS AAGY]_(n) SLP L3.0 [(GAGAGS)₉ GAA PGASIKVAVSAGPS AGY]_(n)SLP L3.1 [(GAGAGS)₉ GAA PGASIKVAVSGPS AGY]_(n) SLP F9 [(GAGAGS)₉RYVVLPRPVCFEK AAGY]_(n) ELP I [(VPGVG)₄]_(n) SELP 0 [(GVGVP)₈(GAGAGS)₂]_(n) SELP 1 [GAA(VPGVG)₄ VAAGY (GAGAGS)₉]_(n) SELP 2[(GAGAGS)₆ GAAGY (GAGAGS)₅ (GVGVP)₈]_(n) SELP 3 [(GVGVP)₈ (GAGAGS)₈]_(n)SELP 4 [(GVGVP)₁₂ (GAGAGS)₈]_(n) SELP 5 [(GVGVP)₁₆ (GAGAGS)₈]_(n) SELP 6[(GVGVP)₃₂ (GAGAGS)₈]_(n) SELP 7 [(GVGVP)₈ (GAGAGS)₆]_(n) SELP 8[(GVGVP)₈ (GAGAGS)₄]_(n) KLP 1.2 [(AKLKLAEAKLELAE)₄]_(n) CLP 1[GAP(GPP)₄]_(n) CLP 2 {[GAP(GPP)₄]₂ [GPAGPVGSP}_(n) CLP-CB {[GAP(GPP)₄]₂(GLPGPKGDRGDAGPKGADGSPGPA) GPAGPVGSP}_(n) CLP 3 (GAPGAPGSQGAPGLQ)_(n)Repetitive amino acid sequences of selected protein polymers. SLP = silklike protein; SLPF = SLP containing the RGD sequence from fibronectin;SLPL 3/0 and SLPL 3/1 = SLP containing two difference sequences fromlaminin protein; ELP = elastin like protein; SELP = silk elastin likeprotein; CLP = collagen like protein; CLP-CB = CLP containing a cellbinding domain from human collagen; KLP = keratin like proteinThe nature of the elastinlike blocks, and their length and positionwithin the monomers influences the water solubility of the SELPpolymers. For example, decreasing the length and/or content of thesilklike block domains, while maintaining the length of the elastinlikeblock domains, increases the water solubility of the polymers. For amore detailed discussion of the production of SLP's, ELP's, CLP's,SLPF's and SELP's as well as their properties and characteristics see,for example, in J. Cappello et al., Biotechnol. Prog., 6, 198 (1990),the full disclosure of which is incorporated by reference herein. Onepreferred SELP, SELP7, has an elastin:silk ratio of 1.33, and has 45%silklike protein material and is believed to have weight averagemolecular weight of 80,338.

The amount of the biocompatible protein component utilized in thecoatable composition will be dependent upon the amount of coatablecomposition desired in relation to the other components of the deviceand the particular biocompatible protein component chosen for use in thecoatable composition. Furthermore, the amount of coatable compositionutilized in the coating of the film will be determinative of the size ofthe film, and thus, the size of the cohesive body and the resultingprotein matrix device. That is, inasmuch as the amounts of the remainingcomponents are dependent upon the amount of biocompatible proteincomponent utilized, the amount of biocompatible protein component may bechosen based upon the aforementioned parameters.

Any biocompatible solvent may be utilized in the method andcorresponding protein matrix device of the present invention. By using abiocompatible solvent, the risk of adverse tissue reactions to residualsolvent remaining in the device after manufacture is minimized.Additionally, the use of a biocompatible solvent reduces the potentialstructural and/or pharmacological degradation of the pharmacologicallyactive agent that some such pharmacologically active agents undergo whenexposed to organic solvents. Suitable biocompatible solvents for use inthe method of the present invention include, but are not limited to,water; dimethyl sulfoxide (DMSO); biocompatible alcohols, such asmethanol and ethanol; various acids, such as formic acid; oils, such asolive oil, peanut oil and the like; ethylene glycol, glycols; andcombinations of these and the like. Preferably, the biocompatiblesolvent comprises water. The amount of biocompatible solvent utilized inthe coatable composition will preferably be that amount sufficient toresult in the composition being fluid and flowable enough to becoatable. Generally, the amount of biocompatible solvent suitable foruse in the method of the present invention will range from about 50% toabout 500%, preferably from about 100% to about 300% by weight, basedupon the weight of the biodegradable polymeric material.

In addition to the biocompatible protein material(s) and thebiocompatible solvent(s), the protein matrix devices of the presentinvention may optionally comprise one or more pharmacologically activeagents. As used herein, “pharmacologically active agent” generallyrefers to a pharmacologically active agent having a direct or indirectbeneficial therapeutic effect upon introduction into a host.Pharmacologically active agents further includes neutraceuticals. Thephrase “pharmacologically active agent” is also meant to indicateprodrug forms thereof. A “prodrug form” of a pharmacologically activeagent means a structurally related compound or derivative of thepharmacologically active agent which, when administered to a host isconverted into the desired pharmacologically active agent. A prodrugform may have little or none of the desired pharmacological activityexhibited by the pharmacologically active agent to which it isconverted. Representative examples of pharmacologically active agentsthat may be suitable for use in the protein matrix device of the presentinvention include, but are not limited to, (grouped by therapeuticclass):

Antidiarrhoeals such as diphenoxylate, loperamide and hyoscyamine;

Antihypertensives such as hydralazine, minoxidil, captopril, enalapril,clonidine, prazosin, debrisoquine, diazoxide, guanethidine, methyldopa,reserpine, trimethaphan;

Calcium channel blockers such as diltiazem, felodipine, amodipine,nitrendipine, nifedipine and verapamil;

Antiarrhyrthmics such as amiodarone, flecamide, disopyramide,procainamide, mexiletene and quinidine,

Antiangina agents such as glyceryl trinitrate, erythrityl tetranitrate,pentaerythritol tetranitrate, mannitol hexanitrate, perhexylene,isosorbide dinitrate and nicorandil;

Beta-adrenergic blocking agents such as alprenolol, atenolol,bupranolol, carteolol, labetalol, metoprolol, nadolol, nadoxolol,oxprenolol, pindolol, propranolol, sotalol, timolol and timolol maleate;

Cardiotonic glycosides such as digoxin and other cardiac glycosides andtheophylline derivatives;

Adrenergic stimulants such as adrenaline, ephedrine, fenoterol,isoprenaline, orciprenaline, rimeterol, salbutamol, salmeterol,terbutaline, dobutamine, phenylephrine, phenylpropanolamine,pseudoephedrine and dopamine;

Vasodilators such as cyclandelate, isoxsuprine, papaverine,dipyrimadole, isosorbide dinitrate, phentolamine, nicotinyl alcohol,co-dergocrine, nicotinic acid, glycerl trinitrate, pentaerythritoltetranitrate and xanthinol;

Antimigraine preparations such as ergotanmine, dihydroergotamine,methysergide, pizotifen and sumatriptan;

Anticoagulants and thrombolytic agents such as warfarin, dicoumarol, lowmolecular weight hepafins such as enoxaparin, streptokinase and itsactive derivatives;

Hemostatic agents such as aprotinin, tranexarnic acid and protamine;

Analgesics and antipyretics including the opioid analgesics such asbuprenorphine, dextromoramide, dextropropoxyphene, fentanyl, alfentanil,sufentanil, hydromorphone, methadone, morphine, oxycodone, papavereturn,pentazocine, pethidine, phenopefidine, codeine dihydrocodeine;acetylsalicylic acid (aspirin), paracetamol, and phenazone;

Neurotoxins such as capsaicin;

Hypnotics and sedatives such as the barbiturates amylobarbitone,butobarbitone and pentobarbitone and other hypnotics and sedatives suchas chloral hydrate, chlormethiazole, hydroxyzine and meprobamate;

Antianxiety agents such as the benzodiazepines alprazolam, bromazepam,chlordiazepoxide, clobazam, chlorazepate, diazepam, flunitrazepam,flurazepam, lorazepam, nitrazepam, oxazepam, temazepam and triazolam;

Neuroleptic and antipsychotic drugs such as the phenothiazines,chlorpromazine, flupbenazine, pericyazine, perphenazine, promazine,thiopropazate, thioridazine, trifluoperazine; and butyrophenone,droperidol and haloperidol; and other antipsychotic drugs such aspimozide, thiothixene and lithium;

Antidepressants such as the tricyclic antidepressants amitryptyline,clomipramine, desipramine, dothiepin, doxepin, imipramine,nortriptyline, opipramol, protriptyline and trimipramine and thetetracyclic antidepressants such as mianserin and the monoamine oxidaseinhibitors such as isocarboxazid, phenelizine, tranylcypromine andmoclobemide and selective serotonin re-uptake inhibitors such asfluoxetine, paroxetine, citalopram, fluvoxamine and sertraline;

CNS stimulants such as caffeine and 3-(2-aminobutyl)indole;

Anti-alzheimer's agents such as tacrine;

Anti-Parkinson's agents such as amantadine, benserazide, carbidopa,levodopa, benztropine, bipefiden, benzhexyl, procyclidine and dopamine-2agonists such as S(−)-2-(N-propyl-N-2-thienyl ethylamino)-5-hydroxytetralin (N-0923)-,

Anticonvulsants such as phenyloin, valproic acid, primidone,phenobarbitone, methylphenobarbitone and carbamazepine, ethosuximide,methsuximide, phensuximide, sulthiame and clonazepam,

Antiemetics and antinauseants such as the phenothiazinesprochloperazine, thiethylperazine and 5HT-3 receptor antagonists such asondansetron and granisetron, as well as dimenhydrinate, diphenhydramine,metoclopramide, domperidone, hyoscine, hyoscine hydrobromide, hyoscinehydrochloride, clebopride and brompride;

Non-steroidal anti-inflammatory agents including their racemic mixturesor individual enantiomers where applicable, preferably which can beformulated in combination with dermal penetration enhancers, such asibuprofen, flurbiprofen, ketoprofen, aclofenac, diclofenac, aloxiprin,aproxen, aspirin, diflunisal, fenoprofen, indomethacin, mefenamic acid,naproxen, phenylbutazone, piroxicam, salicylamide, salicylic acid,sulindac, desoxysulindac, tenoxicam, tramadol, ketoralac, flufenisal,salsalate, triethanolamine salicylate, atninopyrine, antipyrine,oxyphenbutazone, apazone, cintazone, flufenamic acid, clonixerl,clonixin, meclofenamic acid, flunixin, colchicine, demecolcine,allopurinol, oxypurinol, benzydamine hydrochloride, dimefadane,indoxole, intrazole, mimbane hydrochloride, paranylene hydrochloride,tetrydamine, benzindopyrine hydrochloride, fluprofen, ibufenac,naproxol, fenbufen, cinchophen, diflumidone sodium, fenamole, flutiazin,metazamide, letimide hydrochloride, nexeridine hydrochloride,octazamide, molinazole, neocinchophen, nimazole, proxazole citrate,tesicam, tesimide, tolmetin, and triflumidate;

Antirheumatoid agents such as penicillamine, aurothioglucose, sodiumaurothiomalate, methotrexate and auranofin;

Muscle relaxants such as baclofen, diazepam, cyclobenzaprinehydrochloride, dantrolene, methocarbamol, orphenadrine and quinine;

Agents used in gout and hyperuricaemia such as allopurinol, colchicine,probenecid and sulphinpyrazone;

Oestrogens such as oestradiol, oestriol, oestrone, ethinyloestradiol,mestranol, stilboestrol, dienoestrol, epioestriol, estropipate andzeranol;

Progesterone and other progestagens such as allyloestrenol,dydrgesterone, lynoestrenol, norgestrel, norethyndrel, norethisterone,norethisterone acetate, gestodene, levonorgestrel, medroxyprogesteroneand megestrol;

Antiandrogens such as cyproterone acetate and danazol;

Antioestrogens such as tamoxifen and epitiostanol and the aromataseinhibitors, exemestane and 4-hydroxy-androstenedione and itsderivatives;

Androgens and anabolic agents such as testosterone, methyltestosterone,clostebol acetate, drostanolone, furazabol, nandrolone oxandrolone,stanozolol, trenbolone acetate, dihydro-testostero17-(a-methyl-19-noriestosterone and fluoxymesterone;

5-alpha reductase inhibitors such as finastride, turosteride, LY-191704and MK-306-1;

Corticosteroids such as betamethasone, betamethasone valerate,cortisone, dexamethasone, dexamethasone 21-phosphate, fludrocortisone,flumethasone, fluocinonide, fluocinonide desonide, fluocinolone,fluocinolone acetonide, fluocortolone, halcinonide, halopredone,hydrocortisone, hydrocortisone 17-valerate, hydrocortisone 17-butyrate,hydrocortisone 21-acetate, methylprednisolone, prednisolone,prednisolone 21-phosphate, prednisone, triamcinolone, triamcinoloneacetonide;

Glycosylated proteins, proteoglycans, glycosaminoglycans such aschondroitin sulfate; chitin, acetyl-glucosamine, hyaluronic acid;

Complex carbohydrates such as glucans;

Further examples of steroidal anti-inflammatory agents such ascortodoxone, fludroracetonide, fludrocortisone, difluorsone diacetate,flurandrenolone acetonide, medrysone, amcinafel, amcinafide,betamethasone and its other esters, chloroprednisone, clorcortelone,descinolone, desonide, dichlofisone, difluprednate, flucloronide,flumethasone, flunisolide, flucortolone, fluoromethalone, fluperolone,fluprednisolone, meprednisone, methylmeprednisolone, paramethasone,cortisone acetate, hydrocortisone cyclopentylpropionate, cortodoxone,flucetonide, fludrocortisone acetate, amcinafal, amcinafide,betamethasone, betamethasone benzoate, chloroprednisone acetate,clocortolone acetate, descinolone acetonide, desoximetasone,dichlorisone acetate, difluprednate, flucloronide, flumethasonepivalate, flunisolide acetate, fluperolone acetate, fluprednisolonevalerate, paramethasone acetate, prednisolamate, prednival,triamcinolone hexacetonide, cortivazol, formocortal and nivazoll;

Pituitary hormones and their active derivatives or analogs such ascorticotrophin, thyrotropin, follicle stimulating hormone (FSH),luteinising hormone (LH) and gonadotrophin releasing hormone (GnRH);

Hypoglycemic agents such as insulin, chlorpropamide, glibenclamide,gliclazide, glipizide, tolazamide, tolbutamide and metformin;

Thyroid hormones such as calcitonin, thyroxine and liothyronine andantithyroid agents such as carbimazole and propylthiouracil;

Other miscellaneous hormone agents such as octreotide;

Pituitary inhibitors such as bromocriptine;

Ovulation inducers such as clomiphene;

Diuretics such as the thiazides, related diuretics and loop diuretics,bendrofluazide, chlorothiazide, chlorthalidone, dopamine,cyclopenthiazide, hydrochlorothiazide, indapamide, mefruside,methycholthiazide, metolazone, quinethazone, bumetanide, ethacrynic acidand frusemide and potasium sparing diuretics, spironolactone, amilorideand triamterene;

Antidiuretics such as desmopressin, lypressin and vasopressin includingtheir active derivatives or analogs;

Obstetric drugs including agents acting on the uterus such asergometfine, oxytocin and gemeprost;

Prostaglandins such as alprostadil (PGEI), prostacyclin (PG12),dinoprost (prostaglandin F2-alpha) and misoprostol;

Antimicrobials including the cephalospofins such as cephalexin,cefoxytin and cephalothin;

Penicillins such as amoxycillin, amoxycillin with clavulanic acid,ampicillin, bacampicillin, benzathine penicillin, benzylpenicillin,carbenicillin, cloxacillin, methicillin, phenethicillin,phenoxymethylpenicillin, flucloxacillin, meziocillin, piperacillin,ticarcillin and azlocillin;

Tetracyclines such as minocycline, chlortetracycline, tetracycline,demeclocycline, doxycycline, methacycline and oxytetracycline and othertetracycline-type antibiotics;

Amnioglycoides such as amikacin, gentamicin, kanamycin, neomycin,netilmicin and tobramycin;

Antifungals such as amorolfine, isoconazole, clotrimazole, econazole,miconazole, nystatin, terbinafine, bifonazole, amphotericin,griseofulvin, ketoconazole, fluconazole and flucytosine, salicylic acid,fezatione, ticlatone, tolnaftate, triacetin, zinc, pyrithione and sodiumpyfithione;

Quinolones such as nalidixic acid, cinoxacin, ciprofloxacin, enoxacinand norfloxacin;

Sulphonamides such as phthalysulphthiazole, sulfadoxine, sulphadiazine,sulphamethizole and sulphamethoxazole;

Sulphones such as dapsone;

Other miscellaneous antibiotics such as chloramphenicol, clindamycin,erythromycin, erythromycin ethyl carbonate, erythromycin estolate,erythromycin glucepate, erythromycin ethylsuccinate, erythromycinlactobionate, roxithromycin, lincomycin, natamycin, nitrofurantoin,spectinomycin, vancomycin, aztreonarn, colistin IV, metronidazole,timidazole, fusidic acid, trimethoprim, and 2-thiopyridine N-oxide;halogen compounds, particularly iodine and iodine compounds such asiodine-PVP complex and diiodohydroxyquin, hexachlorophene;chlorhexidine; chloroan-tine compounds; and benzoylperoxide;

Antituberculosis drugs such as ethambutol, isoniazid, pyrazinamide,rifampicin and clofazimine;

Antimalarials such as primaquine, pyrimethamine, chloroquine,hydroxychloroquine, quinine, mefloquine and halofantrine;

Antiviral agents such as acyclovir and acyclovir prodrugs, famcyclovir,zidovudine, didanosine, stavudine, lamivudine, zalcitabine, saquinavir,indinavir, ritonavir, n-docosanol, tromantadine and idoxuridine;

Anthelmintics such as mebendazole, thiabendazole, niclosamide,praziquantel, pyrantel embonate and diethylcarbamazine;

Cytotoxic agents such as plicamycin, cyclophosphamide, dacarbazine,fluorouracil and its prodrugs (described, for example, in InternationalJournal of Pharmaceutics, 111, 223-233 (1994)), methotrexate,procarbazine, 6-mercaptopurine and mucophenolic acid;

Anorectic and weight reducing agents including dexfenflurarnine,fenfluramine, diethylpropion, mazindol and phentermine;

Agents used in hypercalcaemia such as calcitriol, dihydrotachysterol andtheir active derivatives or analogs;

Antitussives such as ethylmorphine, dextromethorphan and pholcodine;

Expectorants such as carbolcysteine, bromhexine, emetine, quanifesin,ipecacuanha and saponins;

Decongestants such as phenylephrine, phenylpropanolamine andpseudoephedrine; Bronchospasm relaxants such as ephedrine, fenoterol,orciprenaline, rimiterol, salbutamol, sodium cromoglycate, cromoglycicacid and its prodrugs (described, for example, in International Journalof Pharmaceutics 7, 63-75 (1980)), terbutaline, ipratropium bromide,salmeterol and theophylline and theophylline derivatives;

Antihistamines such as meclozine, cyclizine, chlorcyclizine,hydroxyzine, brompheniramine, chlorpheniramine, clemastine,cyproheptadine, dexchlorpheniramine, diphenhydramine, diphenylamine,doxylatnine, mebhydrolin, pheniramine, tripolidine, azatadine,diphenylpyraline, methdilazine, terfenadine, astemizole, loratidine andcetirizine;

Local anaesthetics such as bupivacaine, amethocaine, lignocaine,lidocaine, cinchocaine, dibucaine, mepivacaine, prilocalne, etidocaine,veratridine (specific c-fiber blocker) and procaine;

Stratum corneum lipids, such as ceramides, cholesterol and free fattyacids, for improved skin barrier repair [Man, et al. J. Invest.Dermatol., 106(5), 1096, (1996)];

Neuromuscular blocking agents such as suxamethonium, alcuronium,pancuronium, atracurium, gallamine, tubocurarine and vecuronium;

Smoking cessation agents such as nicotine, bupropion and ibogaine;

Insecticides and other pesticides which are suitable for localapplication;

Dermatological agents, such as vitamins A, C, B1, B2, B6, B12, and E,vitamin E acetate and vitamin E sorbate;

Allergens for desensitisation such as house, dust or mite allergens;

Nutritional agents and neutraceuticals, such as vitamins, essentialamino acids and fats;

acromolecular pharmacologically active agents such as proteins, enzymes,peptides, polysaccharides (such as cellulose, amylose, dextran, chitin),nucleic acids, cells, tissues, and the like; and

Keratolytics such as the alpha-hydroxy acids, glycolic acid andsalicylic acid.

The protein matrix devices as disclosed herein may also be utilized forDNA delivery, either naked DNA, plasma DNA or any size DNA delivery.Also, the protein matrix may be utilized for delivery of RNA types ofsenses, or oligonucleotides that may be man-made portions of DNA or RNA.The protein matrix could also be utilized for delivery of compounds, asexplained anywhere herein, in ovum or in embryos, as the site forimplantation of the protein matrix.

The DNA, RNA or oligonucleotide may be incorporated into the proteinmatrix utilizing the same process of making the protein matrix device asdescribed above. The only difference would be that the pharmacologicalactive agents utilized would be the DNA, RNA, oligonucleotides and othersuch materials. In one example, a cohesive body may be produced bymaking a composition containing one or more biocompatible proteins, oneor more biocompatible solvents and an antisense type material. Ingeneral the complementary strand of a coding sequence of DNA is the cDNAand the complementary strand of mRNA is the antisense RNA. In variousembodiments of the present invention, antisense material delivered by aprotein matrix device of the present invention binds with mRNA, therebypreventing it from making the protein.

Two of the advantages of including DNA, RNA or oligonucleotides in aprotein matrix device is that such a device includes the benefits oflocal drug delivery to target cells and to have a controlled timerelease component so that there is an extended delivery period. Anadditional advantage to delivery of DNA, RNA or oligonucleotidescomponents is that the DNA, RNA or oligonucleotides components can bereleased in a systematic and controlled manner over a long period oftime. For example, when the antisense components bind with RNA, the bodytends to cleave the RNA thereby inhibiting protein production. Thebiological system responds by making more RNA to make proteins. Theprotein matrix device provides delivery of additional antisensecomponents in a location for an extended period of time, therebyblocking the production of the undesired protein. Also thebiocompatibility of the protein matrix material enhances the bindingcharacteristics of the antisense components to their proper bindingsites. Since the protein matrix material can be fabricated or producedto resemble the host tissue, the host cells are able to better interactwith the administered protein matrix device, thereby facilitating thebinding of the complimentary antisense components delivered by theprotein matrix with the DNA and RNA in the host cells.

Additionally, the use of a protein matrix device in an egg or womb couldbe very useful for a number of applications. For example, a vaccine maybe delivered in ova and then released into the animal, such as mammals,birds or reptiles, even after it's born. Also, the introduction ofpharmacologically active agents that could be put in the egg or womb,could be beneficial in that it could inhibit things like bacteria orviral infection of the egg or womb during incubation and promote thehealthy development of a mature animal. For example, it would bepossible for the protein matrix to provide a drug delivery device forgrowth factors, neutraceuticals like vitamins or other agents that wouldhelp in the growth of the animal after it's hatched, or even during thestage when it is unhatched to facilitate the development of that animal.Another example would be the production of livestock, such as domesticanimals like horses, cattle, pigs, sheep, dogs, cats, chickens orturkeys. If domestic animals would get a head start on growth, it mayenhance their body weight, which would have a tremendous impact on theoverall development of the specimen.

Finally, protein matrices may be produced in particulate forms. Theseforms comprise vaccine particles of all types, including proteinparticles containing antigen components that may be made small enough(2-10 μm) to be absorbed by immunogenic cells for enhanced immuneresponse via subcutaneous, intraparetaneal, intravenous, intramuscular,intrathecal, epidural, intraarticular or any other administrationdelivery means.

The protein matrix device in accordance with the present invention, asmentioned hereinabove, may comprise an amount of a neurotoxin as thepharmacologically active agent. Specifically, inasmuch as some cases ofchronic pain are the result of permanent nerve damage, in some instancesit may be desirable to locally deliver an amount of a neurotoxin to theinjured nerve to destroy that portion of the nerve that is the cause ofthe persistent, chronic pain. One example of a neurotoxin suitable foruse in the present invention is capsaicin, as shown in Examples 4 and12, hereinbelow. If a neurotoxin is to be incorporated into the proteinmatrix device of the present invention, it is preferred that it beincorporated in an amount ranging from about 0.001% to about 5%, morepreferably, from about 0.05% to about 1% by weight, based upon theweight of the biocompatible protein component.

The protein matrix device of the present invention is particularlyadvantageous for the encapsulation/incorporation of macromolecularpharmacologically active agents such as proteins, enzymes, peptides,polysaccharides, nucleic acids, cells, tissues, and the like.Immobilization of macromolecular pharmacologically active agents into oronto a protein matrix device can be difficult due to the ease with whichsome of these macromolecular agents denature when exposed to organicsolvents, some constituents present in bodily fluids or to temperaturesappreciably higher than room temperature. However, since the method ofthe present invention, as well as the protein matrix device formed bythe method utilizes biocompatible solvents such as water, DMSO orethanol, and furthermore does not require heating, the risk of thedenaturation of these types of materials is reduced. Furthermore, due tothe size of these macromolecular pharmacologically active agents, theseagents are encapsulated within the protein matrix upon implantation ofprotein matrix devices in accordance with the present invention, andthereby are protected from constituents of bodily fluids that wouldotherwise denature them. Thus, the protein matrix devices of the presentinvention allow these macromolecular agents may exert their therapeuticeffects, while yet protecting them from denaturation or other structuraldegradation.

Examples of cells which can be utilized as the pharmacologically activeagent in the protein matrix device of the present invention includeprimary cultures as well as established cell lines, includingtransformed cells. Examples of these include, but are not limited topancreatic islet cells, human foreskin fibroblasts, Chinese hamsterovary cells, beta cell insulomas, lymphoblastic leukemia cells, mouse3T3 fibroblasts, dopamine secreting ventral mesencephalon cells,neuroblastold cells, adrenal medulla cells, T-cells combinations ofthese, and the like. As can be seen from this partial list, cells of alltypes, including dermal, neural, blood, organ, stem, muscle, glandular,reproductive and immune system cells, as well as cells of all species oforigin, can be encapsulated successfully by this method. Examples ofproteins which can be incorporated into the protein matrix device of thepresent invention include, but are not limited to, hemoglobin,vasporessin, oxytocin, adrenocorticocotrophic hormone, epidermal growthfactor, prolactin, luliberin or luteinising hormone releasing factor,human growth factor, and the like; enzymes such as adenosine deaminase,superoxide dismutase, xanthine oxidase, and the like; enzyme systems;blood clotting factors; clot inhibitors or clot dissolving agents suchas streptokinase and tissue plasminogen activator; antigens forimmunization; hormones; polysaccharides such as heparin;oligonucleotides; bacteria and other microbial microorganisms includingviruses; monoclonal antibodies; vitamins; cofactors; retroviruses forgene therapy, combinations of these and the like.

An efficacious amount of the aforementioned pharmacologically activeagent(s) can easily be determined by those of ordinary skill in the arttaking into consideration such parameters as the particularpharmacologically active agent chosen, the size and weight of thepatient, the desired therapeutic effect, the pharmacokinetics of thechosen pharmacologically active agent, and the like, as well as byreference to well known resources such as Physicians' Desk Reference®:PDR—52 ed (1998)—Medical Economics 1974. In consideration of theseparameters, it has been found that a wide range exists in the amount ofthe pharmacologically active agent(s) capable of being incorporatedinto, and subsequently released from or alternatively allowed to exertthe agent's therapeutic effects from within, the protein matrix device.More specifically, the amount of pharmacologically active agent that maybe incorporated into and then either released from or active from withinthe protein matrix device may range from about 0.001% to about 200%,more preferably, from about 0.05% to about 100%, most preferably fromabout 0.1% to 70%, based on the weight of the biocompatible proteinmaterial.

In addition to the biocompatible protein material(s), the biocompatiblesolvent(s) and pharmacologically active agent(s), the protein matrixdevices of the present invention advantageously may themselvesincorporate other drug delivery devices that would otherwise typicallymigrate away from the desired delivery site and/or are potentiallyundesirably reactive with surrounding bodily fluids or tissues. Suchmigration is undesirable in that the therapeutic effect of thepharmacological agents encapsulated therein may occur away from thedesired site, thus eliminating the advantage of localized delivery. Whena protein matrix device incorporating a migration-vulnerable and/orreactive drug delivery device (hereinafter referred to as a “two-stageprotein matrix device”) is subsequently implanted, themigration-vulnerable and/or reactive drug delivery device(s) is/are heldin place and protected by the two-stage protein matrix device. Moreparticularly, once implanted and/or administered, the pharmacologicallyactive agent is released by the biodegradable material of themigration-vulnerable drug delivery devices as it degrades. Then thepharmacologically active agents diffuse through the protein matrix ofthe two-stage protein matrix device or is released with the degradationof the protein matrix device of the present invention.

Furthermore, the compressed cohesive body of the protein matrix devicereduces, if not prevents, the potential for undesirable reaction withbodily fluids or tissues that may otherwise occur upon implantation of areactive drug delivery device without the protective protein matrixencapsulation. Examples of such drug delivery devices subject tomigration for the delivery site include, but are not limited to,vesicles, e.g., liposomes, lipospheres and microspheres. Vesicles aremade up of microparticles or colloidal carriers composed of lipids,carbohydrates or synthetic polymer matrices and are commonly used inliquid drug delivery devices. Vesicles, for example, have been used todeliver anesthetics using formulations with polylactic acid, lecithin,iophendylate and phosphotidyl choline and cholesterol. For a discussionof the characteristics and efficiency of drug delivery from vesicles,see, e.g., Wakiyama et al., Chem., Pharm. Bull., 30, 3719 (1982) andHaynes et al., Anesthiol, 74, 105 (1991), the entire disclosures ofwhich are incorporated by reference herein.

Liposomes, the most widely studied type of vesicle, can be formulated toinclude a wide variety of compositions and structures that arepotentially non-toxic, biodegradable and non-immunogenic. Furthermore,studies are in progress to create liposomes that release more drug inresponse to changes in their environment, including the presence ofenzymes or polycations or changes in pH. For a review of the propertiesand characteristics of liposomes see, e.g., Langer, Science, 249, 1527(1990); and Langer, Ann. Biomed. Eng., 23, 101 (1995), the entiredisclosures of which are incorporated by reference herein.

Lipospheres are an aqueous microdispersion of water insoluble, sphericalmicroparticles (from about 0.2 to about 100 um in diameter), eachconsisting of a solid core of hydrophobic triglycerides and drugparticles that are embedded with phospholipids on the surface.Lipospheres are disclosed in U.S. Pat. No. 5,188,837, issued to Domb,the disclosure of which is incorporated herein by reference.

Microspheres typically comprise a biodegradable polymer matrixincorporating a drug. Microspheres can be formed by a wide variety oftechniques known to those of skill in the art. Examples of microsphereforming techniques include, but are not limited to, (a) phase separationby emulsification and subsequent organic solvent evaporation (includingcomplex emulsion methods such as oil in water emulsions, water in oilemulsions and water-oil-water emulsions); (b) coacervation-phaseseparation; (c) melt dispersion; (d) interfacial deposition; (e) in situpolymerization; (f) spray drying and spray congealing; (g) airsuspension coating; and (h) pan and spray coating. These methods, aswell as properties and characteristics of microspheres are disclosed in,e.g., U.S. Pat. No. 4,652,441; U.S. Pat. No. 5,100,669; U.S. Pat. No.4,526,938; WO 93/24150; EPA 0258780 A2-U.S. Pat. No. 4,438,253; and U.S.Pat. No. 5,330,768, the entire disclosures of which are incorporated byreference herein.

Inasmuch as the migration-vulnerable and/or reactive drug deliverydevices will desirably further encapsulate a pharmacologically activeagent, the amount of these devices to be utilized in the two-stageprotein matrix device may be determined by the dosage of thepharmacologically active agent, as determined and described hereinabove.Inasmuch as such migration-vulnerable and/or reactive drug deliverydevices represent solid matter that may change the ability of thecoatable composition to be coated, the amount of such devices to beincluded in a two-stage drug delivery device desirably ranges about10,000 to about 1 billion, more preferably ranges from about 1 millionto about 500 million, and most preferably ranges from about 200 millionto about 400 million.

Additionally, the protein matrix devices formed according to the methodof the present invention may optionally comprise one or more additives.Such additives may be utilized, for example, to facilitate theprocessing of the protein matrix devices, to stabilize thepharmacologically active agents, to facilitate the activity of thepharmacologically active agents, or to alter the release characteristicsof the protein matrix device. For example, when the pharmacologicallyactive agent is to be an enzyme, such as xanthine oxidase or superoxidedismutase, the protein matrix device may further comprise an amount ofan enzyme substrate, such as xanthine, to facilitate the action of theenzyme.

Additionally, hydrophobic substances such as lipids can be incorporatedinto the protein matrix device to extend the duration of drug release,while hydrophilic, polar additives, such as salts and amino acids, canbe added to facilitate, i.e., shorten the duration of, drug release.Exemplary hydrophobic substances include lipids, e.g., tristeafin, ethylstearate, phosphotidycholine, polyethylene glycol (PEG); fatty acids,e.g., sebacic acid erucic acid; combinations of these and the like. Aparticularly preferred hydrophobic additive useful to extend the releaseof the pharmacologically active agents comprises a combination of adimer of erucic acid and sebacic acid, wherein the ratio of the dimer oferucic acid to sebacic acid is 1:4. Exemplary hydrophilic additivesuseful to shorten the release duration of the pharmacologically activeagent include but are not limited to, salts, such as sodium chloride;and amino acids, such as glutamine and glycine. If additives are to beincorporated into the coatable composition, they will preferably beincluded in an amount so that the desired result of the additive isexhibited. Generally, the amount of additives may vary between fromabout 0% to about 300%, preferably from about 100% to 200% by weight,based upon the weight of the biocompatible protein material.

Manufacturing protein matrix devices with the method of the presentinvention imparts many advantageous qualities to the resulting proteinmatrix devices. First of all, by compressing the cohesive body in such amanner, the resulting protein matrix device is substantially cohesiveand durable, i.e., with a solvent content of from about 10% to about60%, preferably of from about 30% to about 50%. Thus, administration ofthe protein matrix device is made easy, inasmuch as it may be easilyhandled to be injected or implanted. Furthermore, once implanted, thebiocompatible protein material may absorb water and swell, therebyassisting the protein matrix device to stay substantially in thelocation where it was implanted or injected. Additionally, since theprotein material may be biodegradable and the pharmacologically activeagent is distributed substantially homogeneously therein, the releasekinetics of the pharmacologically active agent are optimized. Indeed,the components and the amounts thereof to be utilized in the proteinmatrix device may be selected so as to optimize the rate of delivery ofthe pharmacologically active agent depending upon the desiredtherapeutic effect and pharmacokinetics of the chosen pharmacologicallyactive agent.

Finally, since biocompatible solvents are used in the manufacture of theprotein matrix devices, the potential for adverse tissue reactions tochemical solvents are reduced, if not substantially precluded. For allof these reasons, protein matrix devices in accordance with the presentinvention may advantageously be used to effect a local therapeuticresult in a patient in need of such treatment. More specifically, theprotein matrix devices of the present invention may be injected,implanted, or administered via oral, as well as nasal, pulmonary,subcutaneous, or any other parenteral mode of delivery. The proteinmatrix device may be delivered to a site within a patient to illicit atherapeutic effect either locally or systemically. Depending on thedesired therapeutic effect, the protein matrix devices may be used toregenerate tissue, repair tissue, replace tissue, and deliver local andsystemic therapeutic effects such as analgesia or anesthesia, oralternatively, may be used to treat specific conditions, such ascoronary artery disease, heart valve failure, cornea trauma, skin woundsand other tissue specific conditions. Protein matrix devices thatinclude pharmacologically active agents may be utilized in instanceswhere long term, sustained, controlled release of pharmacologicallyactive agents is desirable, such as in the treatment of surgical andpost-operative pain, cancer pain, or other conditions requiring chronicpain management.

Furthermore, the protein matrix devices of the present invention mayincorporate multiple pharmacologically active agents, one or more ofwhich may be agents that are effective to suppress an immune and/orinflammatory response. In this regard, the protein matrix devices willdeter, or substantially prevent the encapsulation that typically occurswhen a foreign body is introduced into a host. Such encapsulation couldpotentially have the undesirable effect of limiting the efficacy of theprotein matrix device.

Additionally, one or more polymeric materials may be included in thecoatable composition to add or enhance the features of the proteinmatrix device. For example, one or more polymeric materials thatdegrades slowly may be incorporated into an embodiment of the proteinmatrix device that degrades in order to provide controllable release ofa pharmacologically active agent that is also incorporated into theprotein matrix device. That is, while a protein matrix device thatincludes a relatively fast-degrading protein material without aparticular polymeric material will readily degrade thereby releasingdrug relatively quickly upon insertion or implantation, a protein matrixdevice that includes a particular polymeric material, such aspolyanhydride, will degrade slowly, as well as release thepharmacologically active agent(s) over a longer period of time. Examplesof biodegradable and/or biocompatible polymeric materials suitable foruse in the drug delivery device of the present invention include, butare not limited to epoxies, polyesters, acrylics, nylons, silicones,polyanhydride, polyurethane, polycarbonate, poly(tetrafluoroethylene)(PTFE), polycaprolactone, polyethylene oxide, polyethylene glycol,poly(vinyl chloride), polylactic acid, polyglycolic acid, polypropyleneoxide, poly(alkylene)glycol, polyoxyethylene, sebacic acid, polyvinylalcohol (PVA), 2-hydroxyethyl methacrylate (HEMA), polymethylmethacrylate, 1,3-bis(carboxyphenoxy)propane, lipids,phosphatidylcholine, triglycerides, polyhydroxybutyrate (PHB),polyhydroxyvalerate (PHV), polyethylene oxide) (PEO), poly ortho esters,poly (amino acids), polycynoacrylates, polyphophazenes, polysulfone,polyamine, poly (amido amines), fibrin, graphite, flexiblefluoropolymer, isobutyl-based, isopropyl styrene, vinyl pyrrolidone,cellulose acetate dibutyrate, silicone rubber, copolymers of these, andthe like. Other materials that may be incorporated into the matrix thatare not considered polymers, but provide enhanced features include, butare not limited to, ceramics, bioceramics, glasses bioglasses,glass-ceramics, resin cement, resin fill; more specifically, glassionomer, hydroxyapatite, calcium sulfate, Al₂O₃, tricalcium phosphate,calcium phosphate salts, alginate and carbon. Additional other materialsthat may be incorporated into the matrix included alloys such as,cobalt-based, galvanic-based, stainless steel-based, titanium-based,zirconium oxide, zirconia, aluminum-based, vanadium-based,molybdenum-based, nickel-based, iron-based, or zinc-based (zincphosphate, zinc polycarboxylate).

Embodiments of the protein matrix device may also be crosslinked byreacting the components of the protein matrix with a suitable andbiocompatible crosslinking agent. Crosslinking agents include, but arenot limited to glutaraldehyde, p-Azidobenzolyl Hydazide,N-5-Azido-2-nitrobenzoyloxysuccinimide,4-[p-Azidosalicylamido]butylamine, any other suitable crosslinking agentand any combination thereof. A description and list of variouscrosslinking agents and a disclosure of methods of performingcrosslinking steps with such agents may be found in the Pierce Endogen2001-2002 Catalog which is hereby incorporated by reference.

Furthermore, it is noted that embodiments of the protein matrix deviceof the present invention may include crosslinking reagents that mayinitiated and thereby perform the crosslinking process by UV lightactivation or other radiation source, such as ultrasound or gamma ray orany other activation means.

The protein matrix may be crosslinked by utilizing methods generallyknown in the art. For example, a protein matrix may be partially orentirely crosslinked by exposing, contacting and/or incubating theprotein matrix device with a gaseous crosslinking reagent, liquidcrosslinking reagent, light or combination thereof. In one embodiment ofthe present invention a tube be crosslinked on the outside surface byexposing the only the outside surface to a crosslinking reagent, such asglutaraldehyde. Such a matrix has the advantages of including an outerexterior that is very pliable and possesses greater mechanicalcharacteristics, but includes an interior surface that retains higherbiofunctional features. For example, cell growth may be controlled onportions of the protein matrix by exposing such areas to crosslinkingreagents while still having portions of the same protein matrix that arenot crosslinked, and thereby producing biofunctional selective featuresfor the entire protein matrix device. For example crosslinking portionsof the protein matrix may be used to change, modify and/or inhibit cellattachment. It is also noted that the pharmacologically active agent mayalso be crosslinked, bonded and/or chemically and/or physically linkedto protein matrix either partially or in totality such that the surfaceof the protein matrix and/or the interior of the protein matrix islinked to the protein matrix material. For example, glutaraldehyde maycross-link heparin to a single surface of a protein matrix device.

Embodiments of the present invention may include the addition ofreagents to properly pH the resulting protein matrix device and therebyenhance the biocompatible characteristics of the device with the hosttissue of which it is to be administered. When preparing the proteinmatrix device, the pH steps of the biocompatable material andbiocompatable solvent occur prior to the partial drying preparation ofthe cohesive body. The pH steps can be started with the addition ofbiocompatable solvent to the protein material or to the mixture ofprotein material and optional biocompatible materials, or the pH stepscan be started after mixing the material(s) and solvent(s) togetherbefore the cohesive body is formed. The pH steps can include theaddition of drops of 0.05N to 4.0N acid or base to the solvent wettedmaterial until the desired pH is reached as indicated by a pH meter, pHpaper or any pH indicator. More preferably, the addition of drops of0.1N-0.5 N acid or base are used. Although any acid or base may be used,the preferable acids and bases are HCl and NaOH, respectively. If knownamounts of biocompatable material are used it may be possible to addacid or base to adjust the pH when the biocompatable material is firstwetted, thereby allowing wetting and pH adjustments to occur in onestep.

The patient to which the protein matrix device is administered may beany patient in need of a therapeutic treatment. Preferably, the patientis a mammal, reptiles and birds. More preferably, the patient is ahuman. Furthermore, the protein matrix device can be implanted in anylocation to which it is desired to effect a local therapeutic response.For example, the protein matrix device may be administered, applied,sutured, clipped, stapled, gas delivered, injected and/or implantedvaginally, in ova, in utero, in uteral, subcutaneously, near heartvalves, in periodontal pockets, in the eye, in the intracranial space,next to an injured nerve, next to the spinal cord, etc. The presentinvention will now be further described with reference to the followingnon-limiting examples and the following materials and methods wereemployed. It is noted that any additional features presented in otherembodiments described herein may be incorporated into the variousembodiments being described.

Drug Delivery Devices:

As previously suggested, various embodiments of the protein matrixdevice of the present invention may be utilized as drug deliverydevices. A drug delivery device produced and administered as previouslydisclosed or suggested includes the biocompatible features of thecomponents of the protein matrix and thereby reduces or prevents theundesirable effects of toxicity and adverse tissue reactions that may befound in many other types of drug delivery devices. Furthermore, thecontrolled release characteristics of this type of drug delivery deviceprovides for a higher amount of pharmacologically active agent(s) thatmay be incorporated into the matrix. The controlled release of such adrug delivery device is partially attributed to the homogenousdistribution of the pharmacologically active agent(s) throughout thedrug delivery device. This homogenous distribution provides for a moresystematic, sustainable and consistent release of the pharmacologicallyactive agent(s) by gradual degradation of the matrix or diffusion of thepharmacologically active agent(s) out of the matrix. As a result, therelease characteristics of the pharmacologically active agent from theprotein matrix material and/or device are enhanced.

Additionally, the systematic, sustainable and consistent release of thedrug delivery device may be attributed to the cohesive and interactionfeatures present in the drug delivery device. As previously described,the protein matrix is compressed to eliminate part or all of the bulkwater present in the cohesive body. This compression also compels andinfluences additional attracting forces amongst the protein molecules,solvent molecules and pharmacologically active agent molecules includedin the matrix that would not be found if compression was not undertaken.Also other optional biocompatible materials, if included in the matrix,will be compelled and influenced to interact with the pharmacologicallyactive agents to augment their release characteristics. This additionalbinding characteristic provides for a more systematic and controllablerelease of the pharmacologically active agents that are either trappedby interacting protein, optional biocompatible material and solventmolecules or that are also interacting with the protein, optionalbiocompatible material and solvent molecules themselves. Augmentationmay include inhibiting or enhancing the release characteristics of thepharmacologically active agent(s). For example, a multi-layered drugdelivery device may comprise alternating layers of protein matrixmaterial that have sequential inhibiting and enhancing biocompatiblematerials included, thereby providing a pulsing release ofpharmacologically active agents. A specific example may be utilizingglutamine in a layer as an enhancer and polyanhydride as an inhibitor.The inhibiting layer may include drugs or no drugs.

As previously suggested, embodiments of the drug delivery devices,produced and administered utilizing the methods of the presentinvention, are capable of the sustainable, controllable local deliveryof pharmacologically active agent(s), while also providing the advantageof being capable of being degraded, and preferably safely resorbedand/or remodeled into the surrounding host tissue. The resorbablecharacteristic of various embodiments of the present inventioneliminates the need for the removal of the drug delivery device from thepatient once the pharmacologically active agent(s) have been completelydelivered from the matrix. Alternatively, the drug delivery device maybe produced to remain in the patient and provide a systematic andcontrollable diffusion of the pharmacologically active agent(s) asdescribed and suggested previously.

The drug delivery device of present invention may be formed into anyshape and size, such as a cylinder, a tube, a wafer, particles or anyother shape that may optimize the delivery of the incorporatedpharmacologically active agent. For example, the drug delivery devicemay be administered to a patient in the form of particles. FIGS. 4 and 5depict embodiments of the drug delivery device in particulate form.Particles may be produced by pulverizing the protein matrix followingthe freezing of the matrix in liquid nitrogen or by utilizing otherfreeze fracture or particle forming techniques. A characteristic of theprotein particles is that they no longer aggregate when in theparticulate state. The protein matrix in particulate form may beadministered to a patient in many ways, but have the propercharacteristics which allow it to be a very good injectible.Furthermore, cells can be attached to particles and/or may beincorporated into the larger matrix. Any types cells such as eukaryoticcells, organ cells, such as live islets of the pancreas (for productionof insulin) may be included in a particulate drug delivery device.Furthermore, the particles may include a mixture of drugs incorporatedwithin the protein matrix and may be taken orally or through nasalmucosa, wherein the particles may interact with cellular membranesand/or body fluids.

Also, a release mechanism may be included in the protein matrix drugdelivery device for the release of the one or more pharmacologicallyactive agents. The release mechanism may be a material that encapsulatesa larger drug delivery device, such as a cylinder or the releasemechanism may be within a protein matrix material that includesencapsulated particles of either the drug delivery device or particlesof one or more pharmacologically active agents. Additionally, theprotein matrix may also encapsulate an drug delivery device largerand/or different than a particle that is covered by the releasemechanism material.

FIG. 5A depicts and embodiment of a protein matrix device that includesa release mechanism. The release mechanism 40 is positioned within aprotein matrix material 42. Generally, the mechanism 40 is a materialthat creates a shell around the pharmacologically active agents 44 andinhibits their release until opened by some outside stimuli 46.Normally, the pharmacologically active agent can be released by a pulseof energy, radiation or a chemical reagent acting upon the encapsulatingsubstance. For example, a drug delivery device comprising apharmacologically active agent encapsulated in a polyanhydride coatinginhibits release of the pharmacologically active agent and/or itsinteraction with the host tissue. In this example, the pharmacologicallyactive agents can be released when the polyanhydride surface iscontacted with an ultrasound pulse. Such an embodiment has manyadvantages in treating afflictions that may require an extended timeperiod before release of the pharmacologically active agent isnecessary.

Treatment of cancer or chronic pain may be examples of afflictions thatmay benefit from such an embodiment. The retention of chemotherapy drugslocalized in an area of the patient that includes cancerous tissue maybe beneficial to the long term treatment of the patient. The treatmentmay include implantation of a drug delivery device that includes arelease mechanism in a position of the body wherein cancerous tissueshas been previously resected. Upon determination that cancerous cellgrowth may be ongoing or occurring again, the drug deliver device can bereleased by some stimuli, such as a ultrasound pulse or chemicalreagent. The stimuli opens the release mechanism material and allows thehost tissue to interact with the pharmacologically active agents.

Encapsulated or Coated Stent Devices:

Other embodiments of the present invention include the utilization ofthe protein matrix material in encapsulated or coated stent devices. Astent is a tube made of metal or plastic that is inserted into a vesselor passage to keep the lumen open and prevent closure due to a strictureor external compression. Stents are commonly used to keep blood vesselsopen in the coronary arteries, into the oesophagus for strictures orcancer, the ureter to maintain drainage from the kidneys, or the bileduct for pancreatic cancer or cholangiocarcinoma. Stents are alsocommonly utilized in other vascular and neural applications to keepblood vessels open and provide structural stability to the vessel.Stents are usually inserted under radiological guidance and can beinserted percutaneously. Stents are commonly made of gold or stainlesssteel. Gold is considered more biocompatible. However, stentsconstructed of any suitable material may be utilized with the proteinmatrix of the present invention.

Encapsulation or coating of a stent with the protein matrix material ofthe present invention produces a device that is more biocompatible withthe host tissue than the stent device alone. Such encapsulation orcoating of the stent reduces or prevents adverse immuno-responsereactions to the stent device being administered and further enhancesacceptance and remodeling of the device by the host tissue. Furthermore,encapsulated or coated stent devices may also include one or morepharmacologically active agents, such as heparin, within or attached tothe protein matrix material that may assist in the facilitation oftissue acceptance and remodeling as well as inhibit additional adverseconditions sometimes related to implantation of stents, such as blockageof the vessel from platelet aggregation. In addition to anti-plateletaggregation drugs, anti-inflammatory agents, gene altering agents suchas antisense, and other pharmacologically active agents can beadministered locally to the host tissue.

The protein matrix material may completely encapsulate or otherwise coatthe exterior of the stent. Generally, the encapsulated or coated stentdevice is made in a similar process as described above. FIG. 6 depicts acompression molding device wherein the inner insert 18 includes amandrel 29 that extends upward from the insert 18 into the chamber 17.Following preparation of the cohesive body 23, inner insert 18 isinserted into the cavity 16. A stent 32 is positioned over the mandrel29 and the cohesive body 22 is placed in the cavity and compressed.Encapsulation or coating of the stent 32 is determined by the size ofthe mandrel 29 utilized in the compression molding device. A stent 32that fits snuggly over the mandrel 29 will allow for only a coating uponthe exterior of the stent 32. A smaller mandrel 29 that does provide asnug fit for the stent 32 will allow protein matrix material to movebetween the mandrel 29 and the stent 32 thereby creating anencapsulation of the stent 32. The encapsulated or coated stent deviceis then removed from the compression molding device in a similar way asdescribed above and shown in FIG. 3. The stent device, eitherencapsulated or coated generally has a wall thickness of approximately0.05 mm to 2 mm and preferably has a wall thickness of 0.15 to 0.50 mm.

As previously described additional polymeric and other biocompatiblematerials may be included in the protein matrix material to provideadditional structural stability and durability to the encapsulated orcoated stent device. Also, other structural materials, such asproteoglycans, can be used in this process to add greater tissueimitation and biocompatibility. The proteoglycans can replace or bemixed with the protein material in the production of the protein matrixmaterial.

Additionally, the protein matrix material included in the encapsulatedor coated stent cover may be cross-linked to provide additionaldesirable features such as the inhibition of cell growth or to provideadditional structural durability and stability. For example the proteinmatrix material of the encapsulated or coated stent device may becrosslinked by contacting the material with a chemical reagent, such asglutaraldehyde, or other type of crosslinking reagent. FIG. 7 depictsvarious views of a tube made of elastin which has been crosslinked bybeing exposed to a 1% solution of glutaraldehyde for 5 minutes.

FIGS. 8 and 9 depict additional embodiments of encapsulated and coatedstents. FIG. 8 depicts an encapsulated stent device including a proteinmatrix material comprising a 1:1 ratio of elastin to albumen (bovineserum albumin). FIG. 8 further depicts the encapsulated stent deviceinserted within a silastic tube. The encapsulated stent device in FIG. 8is further shown being expanded by insertion and expansion of anangioplasty balloon within the interior of the device. Furthermore, thestent device of FIG. 8 illustrates that the protein matrix materialremains engaged to the stent struts and does not separate from the stentafter the stent device is opened by the angioplasty balloon.

Other embodiments of the stent device of the present invention may beproduced by preparing a stent device that includes a ratio of 2:1:2collagen to elastin to albumen, 4:1 collagen to elastin, 1:4:15 heparinto elastin to collagen, 1:4:15 condroitin to elastin to collagen. Eachembodiment depicted in the Figures illustrates the uniform distributionof the protein matrix material around the stent and also depicts thestrength and durability of the stent after expansion by a balloon.

Furthermore, the stent devices can also be used to incorporate peptidesand other materials that have the ability to inhibit cell migration. Adisadvantage of utilizing stents in a vessel is that the expansion ofthe vessel upon insertion of stent weakens the vessel and may allowsmooth muscle cells to enter into the vessels thereby occluding orrestinosing the vessel. Occlusion of the vessel and restinosis can betreated by utilizing the stent device and vessels or tube grafts of thepresent invention. Vessels and tubular grafts will be explained later inthe text of this disclosure. It is important to note that inserting astent with or without drugs can prevent such breakdown and growth ofcells into the diseased or damaged vessel.

Tissue Grafts:

Additional embodiments of the present invention include the utilizationof the protein matrix material in producing tissue grafts such asvessels; tubular grafts like tracheal tubes, bronchial tubes, catheterfunctioning tubes, lung, gastrointestinal segments; clear matrix grafts;valves; cartilage; tendons; ligaments skin; pancreatic implant devices;and other types of tissue that relate to the heart, brain, nerve, spinalcord, nasal, liver, muscle, thyroid, adrenal, pancreas, and surroundingtissue such as connective tissue, pericardium and peritoneum. It isnoted that a tube does not necessarily have to be cylindrical in shape,but is generally found in that configuration.

Vessels and tubular grafts may be synthesized utilizing the proteinmatrix material. Generally, a vessel is a tubular graft made of theprotein matrix material that includes the growth of cells on and/orwithin the matrix. For example, vessels may be produced utilizing theprotein matrix material by growing endothelial cells on the inside ofthe protein matrix tube and smooth muscle cells on the outside of thetube. Alternatively, a multi-layered vessel may be created with two ormore separate tubes, wherein a smaller tube with endothelial cells grownon the inside of the tube is inserted into a larger tube with smoothmuscle cells grown on the outside of the tube. Both tubes may then becrosslinked on the surface that does not include cell growth to addfurther durability and stability to the vessel. Additional tubularlayers may be included in the vessel that may or may not include thegrowth of cells on the surfaces or within the protein matrix. FIG. 10depicts various views of a multi-layer vessel by illustrating themulti-layer vessel the various tubes inserted within each other and alsoside by side. These layers may also contain pharmacologically activeagents and/or more structural components, such as polymeric materials orstents. The layers will generally stay in position through adhesives,fasteners like sutures, cell interaction, pressure fitting,crosslinking, protein matrix intermolecular forces and other layeralignment means and may adhere or may not adhere to each other. It isalso noted that layers that include cell growth may also includepharmacologically active agents.

Once prepared the tubular graft or vessel may be administered to thepatient as a replacement to a damaged vessel or as a scaffolding devicethat can be inserted into or mounted around the damaged vessel. Vasculartubes, known as STUNTS (Support Tube Using New Technology Stent) can beused for placement within a blood vessel. (A support tube without a wirestent that can “stunt” the growth of smooth muscle cells into the lumenof the vessel to prevent restenosis.) Embodiments of the tubular graftshave form memory and will reform if cut or severed back to its originalform and shape. FIG. 11 depicts an embodiment of the present inventionthat illustrates the capability, compliancy and capacity of the proteinmatrix material to accept sutures and reform to its original shape.

A vessel structure of the present invention will meet the mechanical andhistological requirements of a blood vessel, while providing thebiological and biochemical functions that are necessary for its success.One embodiment that ensures mechanical integrity and biologicalcompatibility is a scaffold comprising collagen and elastin. Theseproteins are the primary components of a typical arterial wall. Thiswill create the natural environment for the endothelial cells, whileproviding the structural characteristics of these proteins.Endothelialization of the cylindrical matrices will provide the criticalhemocompatibility, while also providing the thrombolyticcharacteristics. This feature will allow for the creation ofsmall-diameter vascular grafts with a reduction in thrombosis.Embodiments of the tubular structure will have a diameter ofapproximately 2-4 mm due to the small-diameters of native coronaryarteries. Due to the prevalence of coronary disease and the need foreffective treatments, the proposed tubular structure would be embracedas a compatible vascular graft.

Additionally, the tubular grafts prepared by using the methods of thepresent invention can provide the similar function as the previouslydescribed encapsulated or coated stent devices. The difference betweenthe tubes and the stent device would be the elimination of the stent.The tubes of the present invention have been shown to provide sufficientstrength and durability and may be utilized as a scaffolding in diseasedvessels thereby inhibiting the narrowing of vessels in all regions ofthe patient, such as the cardiovascular and neural regions. The vesselsor tubular grafts may also be inserted under radiological guidance andcan be inserted percutaneously. Similar to the encapsulated or coatedstent devices, the vessels or tubular grafts that include the proteinmatrix material of the present invention are biocompatible and reduce orprevent immunogenicity with the host tissue. Additionally, since thevessels or tubular grafts of the present invention are produced with abiocompatible protein matrix material and may include the growth ofcells from the patient or compatible cells, the vessel or tubular graftadministered to the host tissue further enhances acceptance andremodeling of the vessel or tubular graft by the host tissue. It isagain noted that remodeling of the protein matrix device of the presentinvention is the modifying, adapting and/or transforming the device intoan interwoven and/or functioning part of the host tissue.

Furthermore, the vessels and/or tubular grafts may also include one ormore pharmacologically active agents within or attached to the proteinmatrix material that may assist in the facilitation of tissue acceptanceand remodeling, as well as inhibit additional adverse conditionssometimes related to implantation of vessels, such as plateletaggregation causing blockage of the vessel. In addition to antiplateletaggregation drugs, anti-inflammatory agent, gene altering agents,enzymes, growth factors and other additional pharmacologically activeagents can be included in the vessel and/or tubular graft for localizedadministration to or near the host tissue.

Embodiments of the protein matrix vessels and/or tubular grafts may beprepared by methods similar to those described and suggested above.FIGS. 12 and 13 depict a compression molding device wherein the innerinsert 18 includes a mandrel 29 that extends upward from the insert 18into the chamber 17. FIG. 13 depicts a top view of the compressionmolding device without the upper insert 19 or plunger 14. Following theinsertion of a sufficient amount of cohesive body 22 the upper insert 19and plunger 14 are applied to the cohesive body 22. As with the previouscompression molding device embodiments the pressure applied by theplunger 14 and surfaces of the chamber 17 and mandrel 26 to the cohesivebody 23 removes the bulk water within the cohesive body 23 therebyresulting in the protein matrix device. The vessel and/or tubular graftis then removed from the compression molding device in a similar way asdescribed above and shown in FIG. 3. The vessel and/or tubular graftgenerally has a wall thickness of approximately 0.05 mm to 1 cm andpreferably has a wall thickness of 0.15 to 0.50 mm.

Furthermore, other tissue grafts may be made by including in thecompression molding device a cavity 16 and inserts 18 and 19 that areconfigured to produce the size and shape of the tissue graft desired.For example valves such as heart valves; bone; cartilage; tendons;ligaments skin; pancreatic implant devices; and other types repairs fortissue that relate to the heart, brain, abdomen, breast, palate, nerve,spinal cord, nasal, liver, muscle, thyroid, adrenal, pancreas, andsurrounding tissue such as connective tissue, pericardium and peritoneummay be produced by forming the cavity 16 and inserts 18 and 19 of themolding compression chamber into the corresponding size and shape of theparticular tissue part. It is noted, that the above mentioned tissueparts may optionally include one or more pharmacologically active agentsor other structural materials, such as metal, polymeric and/orbiocompatible materials including wire, ceramic, nylon or polymericmeshes.

As previously described additional polymeric and other biocompatiblematerials may be included in the protein matrix material of the tissuegrafts to provide additional structural stability and durability. Also,other structural materials, such as proteoglycans, can be used in thisprocess. The proteoglycans can be mixed with one or more proteinmaterials in the production of tissue grafts.

Additionally, the protein matrix material included in the tissue graftsmay be cross-linked to provide additional desirable features such as toinhibit cell growth, reduce immunogenicity or provide additionalstructural durability and stability. For example the protein matrixmaterial of the vessels or tubular grafts may be crosslinked bycontacting the material with a chemical reagent, such as glutaraldehyde,or other type of crosslinking reagent similar to the procedure performedon the stent device of FIG. 7.

In another embodiment of the present invention, vessels can be used tobring blood to cell-support constructs made of the protein matrixmaterial and bring the blood acted on by these cells back into thebody's circulation. The cell support constructs provides the biologicalenvironment for the growth and maintenance of various cell types e.g. aprotein matrix cell scaffold for hepatocytes or islet cells can beplaced in a direct blood link. Such a device will provide thehepatocytes or islet cells with adequate access to the blood supply. Forexample, the cell support construct can act similar to a functioningpancreas, liver or other viable organ in a biological system. In otherwords a cell support construct can be produced and incorporated within abiological system as an organ or partial organ replacement.

Another embodiment of the present invention is a protein matrix devicethat is clear. The procedure for making a clear protein matrix comprisesmaking a mold of collagen and/or elastin as described herein and puttingit through a spinning process that aligns the fibers. The clear proteinmatrix may be utilized in cornea transplants. More, specifically, theprocedure includes putting a protein matrix material inside a devicethat spins upon its axis, similar to a nuclear magnetic resonance or NMRtype machine. The spinning device will spin this material at a very highrate around its own axis so that the center of the protein matrix isthrown outward so that the fibers and/or molecules of the protein matrixare aligned.

Since the protein matrix contains water, the protein matrix, at thishigh rate of spin, starts to act like a fluid and slowly moves theprotein matrix molecules into alignment. The greater amounts of waterincorporated into the matrix, the easier to align the protein and theother molecules. The process may be enhanced if other molecules, such asproteoglycans like heparin, are incorporated in the matrix to make theprotein fibers more slippery. As previously mentioned a clear material,such as this, could be used as a cornea transplant upon growing therequisite cells on the clear matrix.

In preparation of a clear protein matrix material, a sample of proteinmatrix material, as prepared by the methods described or suggestedabove, was placed in a probe and inserted into an NMR device. Onceinside the NMR machine the protein matrix is spun for 48-72 hours,thereby aligning the fibers and/or molecules and producing the clearmatrix.

In another embodiment of making the clear protein matrix material it maybe possible to create a device that spins on its axis for this process.The NMR is just spinning the protein matrix around its own axis, so it'spossible to create such a device wherein the protein matrix may beplaced in the center of the spinning device so that it also would spinon its own axis and create the alignment of the fibers and/or moleculesof the protein matrix material.

The protein matrix utilized for making a clear protein matrix could beany shape or size. However, if you're spinning the protein matrix aroundits own axis, more homogenous force may be applied to all parts of thematrix if it were circular or cylindrical. Furthermore if the circle wasmade big enough, it could then be cut out into any shape and size, withthe idea that all parts of that shape received the same kind of forcewhen produced.

Also, the protein matrix material contains water, typically somewherebetween 10-60% water depending upon how it's made. At this high rate ofspin, it is possible to get some flow of material and provide forcesbetween the protein molecules that make them correspond to each other ina certain way. Moreover, this water environment gives them a lot ofmotion and the spinning gets that motion to align so that when you'redone, the fibers align. This alignment produces a clear protein materialmuch like the cornea.

Wound Healing Devices:

Other embodiments of the present invention include wound healing devicesthat utilize the protein matrix material. The wound healing devices maybe configured in any shape and size to accommodate the wound beingtreated. Moreover, the wound healing devices of the present inventionmay be produced in whatever shape and size is necessary to provideoptimum treatment to the wound. These devices can be produced in theforms that include, but are not limited to, plugs, meshes, strips,sutures, or any other form able to accommodate and assist in the repairof a wound. The damaged portions of the patient that may be treated witha device made of the protein matrix material include skin, tissue(nerve, brain, spinal cord, heart, lung, etc.) and bone. Moreover, thewound healing device of the present invention may be configured andformed into devices that include, but are not limited to, dental plugsand inserts, skin dressings and bandages, bone inserts, tissue plugs andinserts, vertebrae, vertebral discs, joints (e.g., finger, toe, knee,hip, elbow, wrist), tissue plugs to close off airway, (e.g., bronchialairway from resected tissue site), other similar devices administered toassist in the treatment repair and remodeling of the damaged tissueand/or bone.

In one embodiment of the wound healing device of the present invention,a protein matrix material may be formed into a dressing or bandage, tobe applied to a wound that has penetrated the skin, that utilizes a verythin amount of protein matrix material. FIG. 14 depicts an ultra-thincollagen/elastin matrix that is approximately 0.1 mm in thickness. Thinmatrices may be made of one or more suitable biocompatible proteinmaterials, one or more biocompatible solvents and optionally one or morepharmacologically active agents. Furthermore, the protein matrixmaterials formed into a thin dressing or bandage may be approximately0.05-5 mm in thickness.

The protein matrix, upon application, adheres to the skin and willremain for days depending upon the conditions. If protected, embodimentsof the protein matrix dressing will remain on the skin for aconsiderable period of time. Moreover, if the protein matrix is actingas a wound dressing and therefore interacting with a wound it will stickvery tightly. The protein matrix is also acts as an adhesive when wetand as it dries. It is also noted that the protein matrix of the presentinvention incorporated into a wound dressing would help facilitate orlessen scarring by helping to close the wound. Furthermore, proteinmatrix dressings or bandages may be prepared to administer beneficiallyhealing and repairing pharmacologically active agents, as well as, actas a device that may be incorporated and remodeled into the repairingtissue of the wound.

In another embodiment of the present invention, the protein matrix canalso be protected with a tape barrier that is put over the matrix andover the wound. A plastic and/or cellophane-like section of material maybe used as a tape barrier that does not stick to the protein matrixmaterial but holds it in place and provides more protection from theenvironment. Tape barriers that are utilized in bandages existing in theart may be used with the dressing of the present invention.

FIG. 15 depicts a wound dressing comprising a protein matrix that ispositioned in the center of a non-adhesive strip of material attached totwo adhesive ends. The protein matrix can be made from a number ofdifferent protein materials including, but not limited to, acollagen/elastin protein mixture (4:1; 4 parts collagen, 1 partelastin). In one embodiment the elastin utilized may be an insolubleelastin made soluble using DMSO. However, a soluble elastin could beused as well. Either type of elastin works well, however, the insolubleis a much cheaper raw material, and it may have some advantages, such asgreater potential matrix strength due to it's insoluble characteristics.

Embodiments of the protein matrix wound healing device, also provide adevice wherein pharmacologically active agents can be impregnated intoit. The matrix or wound dressing may include, but are not limited to,substances that help clotting, such as clotting factors, substanceswhich are helpful for wound healing, such as vitamin E, as well as,anti-bacterial or anti-fungal agents to reduce the chance of infection.Other groups of pharmacologically active agents that may be delivered bythe protein matrix wound dressing are analgesics, local anesthetics,other therapeutics to reduce pain, reduce scarring, reduce edema, and/orother type of drugs that would have very specific effects in theperiphery and facilitate healing.

The inclusion of such pharmacologically active agents in the proteinmatrix dressing also facilitates the controlled release of substances,which would assist in healing and/or treat and prevent infection.Furthermore, the protein matrix interacts with the cells that migrate tothe wound to facilitate the healing process and that require a matrixand/or blood clotting before they can actually start working to closeand remodel the wound area.

The collagen/elastin matrix is made very similar to the cylinders of theprotein matrix drug delivery devices explained in the presentapplication, except that only enough material is utilized to produce athin wafer. Pressure is placed upon this material to flatten it out.Examples of the wound dressings have produced wafers of approximately0.1 mm in thickness. Because insoluble elastin is present in theproduction of the protein matrix a solvent is utilized. Examples ofsolvents utilized in this process are DMSO and ethanol. The insolubleelastin is mixed into the collagen with a judicious amount of solvent tomake the protein matrix.

An embodiment of the present invention utilizes DMSO as the solvent.DMSO has some properties, which provide some benefits. However, anysolvent, which dissolves or sufficiently wets the insoluble elastin maybe used in the present invention. The properties that DMSO has are thatit actually was used for some time by athletes to help relax muscletissue. Athletes utilized DMSO after a long day of working out orplaying in competitions; rubbing it on the skin over the muscle tissuethat was bothering them would relieve the pain from their muscle tissue.DMSO is inexpensive to make and purchase. Additional advantages of usingDMSO in the present invention are that it may assist in the reduction ofmuscle pain that might occur, depending on the location and type of thewound and it also may allow for the use of proteins that are veryinsoluble in a water environment, but assists in the production of astrong protein matrix wound dressing.

Another feature of the wound dressing is that only the part of theprotein matrix dressing that is needed will integrate with the cells ofthe wound and be utilized. Generally, over a period of time, a woundwill remodel and close utilizing only the amount of the protein matrixmaterial necessary to assist in the process. Any remaining proteinmatrix not utilized in the mending of the wound will flake away insimilar fashion as the way dead skin, surrounding and covering thehealed wound, dries and flakes off.

The protein matrix wound dressing could also help people who requiremore assistance than normal for a wound to actually close. Individualswho have problems with wound healing may find that their wound takeslonger to close due to their wound not being able to develop a clotand/or set up a matrix for cells to close the wound. In thesesituations, such as a person with diabetics or ulcers, the proteinmatrix may be utilized to assist in healing. The protein matrix providesa material that assists the wound in closing, especially if clottingfactors and maybe some other factors that are known in the art and areimportant to wound care are incorporated into the protein matrix.

Again, the incorporation into the protein matrix of substances, such asbiochemicals, that would naturally be incorporated into the wound duringhealing may be of benefit in the healing process. The protein matrixitself comes in contact with the wound and supplies a scaffold for thecells to interact with and thereby assists in healing the wound.Therefore, the incorporation of the previously mentioned biochemicals,which can be uniformly dispersed and impregnated into the matrix, canfurther assist in the healing process and increase the prevention ofinfection, reduction of pain, remodeling of the damaged tissue and allother overall healing results.

The biochemicals, previously referred to, such as factor 14, factor 8and other similar biochemicals are most crucial to the beginning stepsof wound care. The impregnation of such biochemicals into a proteinmatrix will translate to a faster closing process and hence a fasterhealing process. These biochemicals are present in our blood at alltimes and are immediately prepared to function when they come in contactwith a wound site. However, sometimes for various reasons a patient'sblood does not have enough of these biochemicals or cannotsatisfactorily supply a sufficient amount to effectively repair a wound.Therefore, the application of a protein matrix as described herein whichis impregnated with such biochemicals can have a beneficial role instimulating and enhancing the healing process.

It is also possible to extend delivery of chemicals or drugs using thisprotein matrix as a wound dressing. In one embodiment this can beaccomplished by providing a protein matrix wound dressing that includesa patch delivery system adjoined immediately behind the protein matrixdressing. In this example a strip, wrap or patch that includes a largerdosage of the chemical or pharmaceutical active component may be appliedbehind the protein matrix not in immediate contact with the wound. Byadministering such a wound healing device, the delivery of chemicalsand/or pharmaceuticals could be extended until the wound was healed orthe desired amount of chemicals and/or pharmaceuticals were applied. Inapplication, the protein matrix would continue to absorb more chemicalsand/or pharmaceuticals from the patch as the initial materialimpregnated in the matrix was being utilized in the wound. Therefore,the protein matrix would provide a controlled release of the chemicaland/or pharmaceutical component and would prevent the administration oftoo much chemical and/or pharmaceutical component from entering apatient's wound prematurely. Additionally, the protein matrix withadjoining patch may be very beneficial for patients who are compromisedin some way from internally supplying the biological substances neededto reduce or prevent them from healing quickly. Examples of suchsituations where such a protein matrix wound healing device would bebeneficial are in cases of diabetes, hemophilia, other clotting problemsor any other type affliction that inhibits the adequate healing of awound. Furthermore, individuals with such conditions may require a greatdeal more than the clotting agents that can be incorporated into a thinprotein matrix. Therefore, the patch may contain more than oneadditional chemical and/or pharmaceutical components that may benefitfrom extended contact with the wound in the healing process.

Additionally, embodiments of a moistened protein matrix dressing thatincludes a patch may be configured to allow a varying controlled releaseof pharmaceuticals through the matrix by providing a matrix that releasemolecules at varying rates based on molecule size. This provides atremendous means for controlling administration of more than onepharmacologically active agent that vary in size. Such controlledrelease facilitates the administration of pharmaceutical molecules intothe wound when they may be needed. For example, the protein matrixdressing may be layered with different types of protein material andbiocompatible polymeric material mixtures that control the release ofmolecules based on size. For example, the protein matrix material mayinclude physical and/or chemical restraints that slow the migration ofvarious size molecules from the patch and through the protein matrixdressing. Furthermore, the larger molecules that are proteins and othermacromolecules that need to be in contact with the wound can beimpregnated into the protein matrix itself.

Furthermore, the protein matrix dressing may be set up with pores thatallow fluid flow through that matrix and also enhances movement of thepharmacologically active agents through the matrix. Pores may be createdin the matrix by incorporating a substance in the cohesive body duringits preparation that may be removed or dissolved out of the matrixbefore administration of the device or shortly after administration.Porosity may be produced in a protein matrix device by the utilizationof materials such as, but not limited to, salts such as NaCl, aminoacids such as glutamine, microorganisms, enzymes, copolymers or othermaterials, which will be leeched out of the protein matrix to createpores. Other functions of porosity are that the pores create leakage sothat cells on outside can receive fluids that include the contents ofthe matrix and also that cells may enter the matrix to interact andremodel the matrix material to better incorporate and function withinthe host tissue.

As described herein a protein matrix may be made porous by theutilization of salts or other such materials. However, it is alsopossible to produce a porous protein matrix by the incorporation of asolution saturated or supersaturated with a gaseous substance, such ascarbon dioxide. In one embodiment, carbonated water may be utilized in asealed and pressurized environment during the production of the proteinmatrix. The utilization of carbonated water creates bubbles within theprotein matrix during the production process. Once the matrix has beenshaped into the desired foam and removed from the sealed and pressurizedenvironment, the gaseous bubbles escape from the matrix leaving a porousmaterial.

Another embodiment for producing a porous protein matrix makes use ofpolyvinyl alcohol (PVA or other water soluble polymers). Polyvinylalcohol (PVA) or other water-soluble polymers can be made into particlesthat correspond to a specific size. The particles are made by firstproducing a gel following standard techniques for that polymer. Forexample, PVA is made into a 4% solution in 100 ml and placed into avacuum oven at 40° C. for 24 hours. The resulting dried gel ispulverized after freezing with liquid nitrogen. The particles are thenseparated by a sieve into specific sizes. The water-soluble polymerparticles are incorporated into the protein matrix so that they can bedissolved by aqueous solutions to provide a protein matrix that is athree dimensional scaffold for cells to migrate and grow within. The PVAparticles will dissolve at rates that are directly proportional to thesize and thickness of the protein matrix. The PVA particles can be madewith cell enhancing agents or chemicals to act as therapeutics so thatresidual particles can facilitate cell migration, growth and/orproliferation from the pore structures.

The protein matrix material of the present invention may also beutilized as port seals for protrusion devices entering and or exitingthe patient. FIG. 16 depicts one embodiment of a protrusion device 34that includes a port seal 36 comprising the protein matrix material ofthe present invention. The port seal 26 may be included around the pointof insertion of a protrusion device, such as an electrical lead or acatheter. Generally, the port seal 36 surrounds the protrusion device 34and insulates it from the host tissue. One or more tabs 38 mayoptionally be included on the port seal 36 to assist in the retention ofthe protrusion device and further seal the opening in the patients skin.The tabs 38 may be inserted under the skin or may remain on the outsideof the patient's skin. Also, the biocompatible seal comprising theprotein matrix material of the present invention provides stability,reduces the seeping of bodily fluid from around the protrusion andreduces or prevents immunogenicity caused by the protrusion device.Furthermore, the port seal may include pharmacologically active agentsthat may be produced to deliver anti-bacterial, analgesic,anti-inflammatory and/or other beneficial pharmacologically activeagents.

Other embodiments of the present invention include wound-healing devicesconfigured and produced as protein matrix biological fasteners, such asthreads, sutures and woven sheets. Threads and sutures comprisingvarious embodiments of the protein matrix material provide abiocompatible fastening and suturing function for temporarily treatingand sealing an open wound. Additionally, the biological fasteners mayinclude pharmacologically active agents that may assist in the healingand remodeling of the tissue within and around the wound.

One method of preparing the biocompatible biological fasteners is tomanufacture sheets of protein matrix material. Once the sheets ofprotein matrix material are prepared each sheet may cut into strips,threads or other shapes to form sutures, threads and other biologicalfasteners (e.g., hemostats). The sheets may be cut using cuttingtechniques known in the art. Also, the protein matrix threads may bewoven into sheets and used as a strengthened protein matrix materialthat has desired porosity. For example, this woven protein matrix mayalso be used with cohesive body to form a protein matrix that has awoven protein matrix encapsulated or filled by protein matrix.

Additionally, fibers (large or small, e.g., macro, micro, nano) of aknown suturing material, such as nylon, may incorporated in the cohesivebody and compressed to make a sheet of protein matrix material. It isnoted that the protein matrix forms a cohesive body around thebiocompatible thread/fibers during compression to encapsulate thebiocompatible fibers into the protein matrix. Once the sheet is preparedit may be cut by methods common to the art to produce a thread/suturethat has biocompatible and durable characteristics.

Additional embodiments of wound healing devices that include the proteinmatrix material of the present invention include but are not limited todental inserts, dental plugs, dental implants, dental adhesives, andother devices utilized for dental applications. Wounds and dentalcomplications, such as dry socket, present within the interior of themouth are generally slow to heal, are painful and/or are susceptible tobacterial and other forms of infection. The dental inserts or implantsof the present invention may be utilized to remedy such problems sincethey are biocompatible with the surrounding host tissue and may bemanufactured to release appropriate pharmacologically active agents thatmay assist in healing, relieve pain and/or reduce bacterial attack ofthe damaged region. Furthermore, the dental plugs, inserts or implantsof the present invention include one or more biocompatible proteinmaterial and one or more biocompatible solvent that may be incorporatedinto and remodeled by the surrounding tissue, thereby hastening thehealing of the damaged region and/or returning the damaged region to itsoriginal state. For example, dental plugs or implants may beadministered to open wounds within the mouth region of the patientfollowing tooth extraction, oral surgery or any other type of injury tothe interior of the mouth to assist in the healing and regeneration ofthe damaged region.

In general, the dental plugs, implants or inserts may be administered tothe damaged area by any method known in the art. For example a dentalplug may be administered to the socket of a tooth after removal byplacing a properly sized and shaped dental plug that includes theprotein matrix of the present invention into the socket. The dental plugmay optionally be fastened to the surrounding tissue of the socket byany means known in the art such as adhesives or sutures. However, it maynot be necessary to use any fastening means since the cells of the hosttissue may be found to readily interact with the plug and begin toincorporate the plug into the host tissue. As previously suggested, sucha dental plug may also include analgesic antibacterial, and otherpharmacologically active agents to reduce or prevent pain and infectionand to promote the reconstruction of the damaged region.

Other Protein Matrix Devices:

The protein matrix material of the present invention may also beutilized in other medical devices to enhance their biocompatibility,provide medical functionality and/or deliver pharmacologically activeagents. One example, of other devices that utilized the protein matrixmaterial of the present invention may be as an intrauterine device(IUD). An IUD is a contraceptive device that is placed within the uterusfor the purpose of inhibiting conception. Generally, the protein matrixmay be produced into any IUD like configuration known in the art andinserted into the uterus. The protein matrix mesh may be prepared byutilizing methods previously described or suggested in the application.Upon insertion of protein matrix mesh and/or particles of any shape intothe uterus, the mesh and/or particles interact with the uterine wallcells to create a natural fibrotic meshwork that closes the uterus byfusing the uterine walls together to thereby inhibit the endometriallining from forming inhibiting menstruation and conception. The IUDprotein matrix device may also include pharmacologically active agentsthat aid in the production of the fibrotic meshwork and/or locally treatthe surrounding tissue.

Additionally other protein matrix device embodiments include a proteinmatrix that has incorporated into it a marker system that allows thematrix to be located and imaged using ultrasound, MRI, X-Ray, PET orother imaging techniques. The image marker can be made with air bubblesor density materials that allow easy visualization of the protein matrixby ultrasound. The incorporated materials can be metallic, gaseous orliquid in nature. Specific materials that may be utilized as imagemarkers incorporated into the protein matrix material include, but arenot limited to, Gd-DPTA. It may be possible to cause the material toreact to an imaging technique, i.e., ultrasound to make bubbles orthrough the addition of another chemical or substance to the system(e.g., peroxide addition to a protein matrix that contains peroxidase asan intrauterine marker that can be monitored by ultrasound). Also, theaddition of a harmless unique salt solution, or enzyme, may promote gasproduction by the protein matrix as an ultrasound maker.

The protein matrix can contain agents that can be seen by ultrasound,MRI, PET, x-ray or any imaging device that is either known, indevelopment or developed in the future.

Other embodiments of the present invention are protein matrices, whichcan include imprints that provide for specific site location forattachment of substances, such as chemicals, cells or enzymes, or forpreventing or reducing attachment of such substances. Examples ofmaterials that may be targeted for specific attachment sites on theprotein matrix may be cell adhesion molecules or electro-conductivemolecules.

The protein matrix can be of any size, shape or form and can beimprinted with any pattern desired depending upon the application. Forexample, an embodiment of the imprinted protein matrix may take the formof a blood vessel. The exterior of the blood vessel may be imprintedwith a pattern that limits the attachment of cellular material thatfacilitates capillary growth to the exterior. This promotion ofangiogenesis provides a number of benefits including the reduction ofinflammation to the vessel surroundings and the further promotion of thesurrounding tissue's acceptance and incorporation of the vessel.

Another embodiment includes the protein matrix in the form of a sphere.Such a matrix may be imprinted in areas with a substance that inhibitsthe binding of biological tissue upon implantation in only thesepredetermined areas. More specifically, a protein matrix may beimpregnated with an adhesive substance, which would facilitate bindingto tissue. Therefore, the portions of the protein matrix imprinted withthe nonbonding substance are thereby prevented from adjoining thesurrounding tissue. However, the regions not imprinted would adhere tothe tissue and perform the intended functions.

Methods of imprinting the protein matrix with a desired pattern can beperformed by any means known in the art. For example, the utilization ofUV light can produce a crosslinking pattern upon the protein matrix.Many difference crosslinking agents can be used but crosslinking agentsthat are only active upon UV activation can selectively attach chemicalsubstances to the protein matrix. This crosslinking can occur either onthe surface or within the protein matrix. One function of such acrosslinking pattern would be to inhibit the attachment of cells.Alternately, it is also possible to attached molecules that will allowattachment of cells. Chemicals, enzymes, short peptides or large peptidesegments can be crosslinked to selected areas of the protein matrix.Such substances can be utilized to attract and enhance the attachmentand/or growth of various cells.

Another embodiment of the present invention relating to an imprintingmethod is the use of masking systems to create the imprinted pattern.The pattern on a protein matrix may be produced by covering the proteinmatrix with a mask that has the desired pattern and exposing the coveredmatrix to a chemical substance, such as glutaraldehyde or anycrosslinking agent (e.g., UV-activated chemical). The chemical substancecontacts the portions of the protein matrix not covered by the mask andcrosslinking occurs. Alternatively, when utilizing UV-activatedchemicals, the mask blocks the light thereby inhibiting crosslinking sothat crosslinking only occurs at unmasked sites. The mask is thenremoved thereby providing a protein matrix with both crosslinked andnon-crosslinked portions. The non-crosslinked areas can providelocations for the attachment or access to chemicals, cells, enzymes,oligonucleotides, other proteins, etc. Furthermore, these site-specificattachment areas of the protein matrix may be utilized for diagnosticreasons, the growth of cells or as access points for other chemicals orenzymes.

Finally the imprinted protein matrix has applications in the proteinchip technology described above. The imprinting of patterns upon theprotein matrix chip may produce chips, which provide a number of similarcharacteristics as a silicon chip or silicon coated substance. Aspreviously suggested, such an embodiment may be beneficial in variousdiagnostic applications.

EXAMPLES

The drug delivery devices of the present invention will now be furtherdescribed with reference to the following non-limiting examples and thefollowing materials and methods that were employed.

Xanthine oxidase, superoxide dismutase, capsaicin and dexamethasone wereobtained from (Sigma Chemical Company, St. Louis Mo.). The silklike,elastinlike polymer SELP7 was obtained from Protein PolymerTechnologies, San Diego, Calif.

Test Method 1. Thermal Sensitivity Test.

The thermal sensitivity tests referred to herein below were conducted asfollows. Thermal sensitivity was measured by the time required for eachrat to withdraw its hind paw from a 56° C. hot plate (commerciallyavailable under the trade designation 35-D from IITC Life ScienceInstruments, Woodland Hills Calif.). Specifically the rats werepositioned to stand with one hind paw on a hot plate and the other on aroom temperature board. Latency to withdraw each hind paw from the hotplate was recorded by alternating paws and allowing at least 15 secondsof recovery between each measurement. If no withdrawal occurred from thehot plate within 15 seconds, the trial was terminated to prevent injuryand the termination time was recorded. Testing ended after threemeasurements per side and the mean was calculated for each side.

Test Method 2. Motor Capacity Test.

The motor capacity tests referred to herein below were conducted asfollows. The rat is held in the same manner as during the thermalsensitivity testing so that it is positioned to stand on one leg againstan electronic balance. The resistance of the rat's leg is measured asthe force against the balance in grams. Previous results from controlexperiments show that a 200-275 gram rat exerts about 150-225 grams offorce with a normal leg. However, if the leg is showing a lack of motorcapacity from local anesthetic action, then forces of only from about 30to about 70 grams are expected. Thus, a lack of motor capacity resultingin the rat exerting only from about 30 to about 70 grams of forceagainst the balance shows that the administered drug delivery device hasdelivered enough of a pharmacologically active agent to produce localanesthetic action.

Example 1 Preparation of a Drug Delivery Device Comprising aBiodegradable Protein and an Enzyme

The enzyme xanthine oxidase was dissolved in deionized water to 0.28units/100 μl. This xanthine oxidase solution was mixed in with 50 mgprotein (SELP 7) to form a coatable composition. The composition wasthen coated on a glass surface to form a film with a thickness of fromabout 0.1 to about 0.3 mm. The coated film was allowed to dry at roomtemperature until dry enough so as to be cohesive, i.e., to a solventcontent of from about 50% to about 70%. The resulting film was rolledup, placed in a 3.5 mm diameter mold and compressed at 1750 psi for 2minutes to form a 3.5 mm diameter cylinder, approximately 5 mm long,utilizing the compression molding device discussed hereinabove. Theresulting cylinder had a solvent content of approximately 30% to about60%. This cylinder was cut into four equal pieces so that each piececontained approximately 0.07 xanthine oxidase units/piece. These pieceswere frozen at −80° C. until used within 4 weeks.

Example 2 Preparation of a Drug Delivery Device Comprising aBiodegradable Protein and an Enzyme

The enzyme superoxide dismutase (SOD) was dissolved in deionized waterto 30.0 units/100 μl. This SOD solution was mixed with 50 mg (SELP7) toform a coatable composition. The composition was then coated on a glasssurface to form a film with a thickness of from about 0.1 mm to about0.3 mm. The coated film was allowed to dry at room temperature until dryenough so as to be cohesive, i.e., to a solvent content of from about50% to about 70%. The resulting film was rolled up, placed in a 3.5 mmdiameter mold and compressed at 1750 psi for 2 minutes to form a 3.5 mmdiameter cylinder, approximately 5 mm long, utilizing the compressionmolding device discussed hereinabove. The resulting cylinder had asolvent content of from about 30% to about 60%. This cylinder was cutinto four equal pieces so that each piece contained approximately 7.5units of SOD per/piece. These pieces were frozen at −80° C. until usedwithin 4 weeks.

Example 3 Preparation of a Drug Delivery Device Comprising aBiodegradable Protein and Lipospheres

Lipospheres with 3.6% of the local anesthetic bupivacaine were made asdescribed in U.S. Pat. No. 5,188,837. From about 200 million to about400 million of these lipospheres were then suspended in 150 μl deionizedwater. This suspension was then mixed with 30 mg SELP7 to form acoatable composition. The composition was then coated onto a glasssurface to form a film with a thickness of from about 0.1 to about 0.3mm. The coated film was allowed to dry at room temperature until thefilm was dry enough so as to be cohesive, i.e., to a solvent content offrom about 50% to about 70%. The resulting film was rolled up, placed ina 3.5 mm diameter mold and compressed at 1750 psi for 2 minutes to forma 3.5 mm diameter cylinder, approximately 4 mm long, utilizing thecompression molding device discussed hereinabove. The resulting cylinderhad a solvent content of from about 30% to about 50%. Four cylinderswere made according to this procedure. These cylinders were refrigeratedat 4° C. until used within 4 weeks.

Example 4 Preparation of a Drug Delivery Device Comprising aBiodegradable Protein and Two Pharmacologically Active Agents

Drug delivery devices were prepared with differing concentrations of thetwo pharmacologically active agents capsaicin and dexamethasone asfollows. Specifically, first drug delivery devices were preparedcomprising 6 mg of capsaicin and 6 mg dexamethasone by dissolving theseamounts in 100 μl ethanol. This solution was then added to a solution of128 mg SELP7 dissolved in 150 μl water to form a coatable composition.This composition was then coated onto a glass surface to form a filmwith a thickness of from about 0.1 mm to about 0.3 mm film. The coatedfilm was allowed to dry at room temperature until dry enough so as to becohesive, i.e., to a solvent content of from about 50% to about 70%. Theresulting film was rolled up, placed in a 3.5 mm diameter mold andcompressed at 7600 psi overnight to form a 3.5 mm diameter cylinder,approximately 5 mm long, utilizing the compression molding devicediscussed. The cylinder was dried to a solvent content of from about 30%to about 50% in a vacuum and then cut into three equal pieces. Frominitially added quantities, each pellet was calculated to containapproximately 2 mg capsaicin and 2 mg dexamethasone, weighingapproximately 35 mg each.

Second drug delivery devices were prepared comprising 6 mg of capsaicinand 1.2 mg dexamethasone by dissolving these amounts of these agents in25 μl ethanol. This solution was then added to a solution of 120 mgSELP7 dissolved in 200 μl deionized water to form a coatablecomposition. This composition was then coated onto two glass surfaces toform two films with thicknesses of from about 0.1 mm to about 0.3 mm.The films were allowed to dry at room temperature until dry enough so asto be cohesive, i.e., to a solvent content of from about 50% to about70%. The resulting films were rolled up, each placed in a 3.5 mmdiameter mold and compressed at 7600 psi overnight to form two 3.5 mmdiameter cylinders, approximately 5 mm long, utilizing the compressionmolding device discussed. The resulting cylinders had a solvent contentof from about 30% to about 60%. These cylinders were cut into 5 equalpellets. From initially added quantities, each pellet was calculated tocontain approximately 2.4 mg capsaicin and 0.24 mg dexamethasone,weighing approximately 30 mg each.

Example 5 Preparation of an Injectable Drug Delivery Device Comprising aBiodegradable Protein, an Additive and an Opioid Analgesic

Injectable drug delivery devices comprising a biodegradable protein, anadditive and an analgesic were made as follows. The opioid analgesic,sufentanil citrate (obtained from National Institute on Drug Abuse) wasdesalted by adding ammonium hydroxide and extracted with n-hexane,collection of solvent and evaporation. The desalted sufentanil wasreconstituted in 20 μl of 90% ethanol containing approximately 4,500,000cpm of tritiated sufentanil (obtained from Jannsen Pharmaceutica,Belgium) to 2.0 mg/20 μl. The biodegradable protein SELP7 was dissolvedin deionized water to 20 mg SELP7/30 μl and spread into a thin layerapproximately 5 cm by 5 cm in area. Immediately thereafter, 10 mg offinely pulverized powder of an additive, fatty acid dimer:sabacic acid(FAD:SA in 1:4 ratio), was added to the center of the protein solutionarea. Immediately thereafter, the sufentanil dissolved in the ethanolwas added very slowly to the mound of FAD:SA over a time period of a fewminutes, i.e., from about 1 to about 5 minutes. After the sufentanilsolution had soaked into the FAD:SA powder, the components werethoroughly mixed to form a coatable composition. The composition wasthen coated onto a glass surface to form a film with a thickness ofapproximately 0.1-0.2 mm. The film was allowed to dry at roomtemperature until capable of forming a cohesive body, i.e., to a solventcontent of from about 50% to about 70%. The resultant was rolled up andcut into many small pieces. Each piece was placed in a 0.63 mm diametermold and compressed at 3,000 psi for 2 minutes to form 0.63 mm diametercylinders, approximately 1.5 mm long and weighing about 0.85 mg to 1.05mg, utilizing the compression molding device discussed hereinabove. Thedrug delivery devices were then exposed to gamma irradiation (60-90KRads) for sterilization and stored in a refrigerator (4° C.) until usedwithin 8 weeks.

Example 6 Preparation of an Implantable Drug Delivery Device Comprisinga Biodegradable Protein, an Additive and an Opioid Analgesic

Implantable drug delivery devices comprising a biodegradable protein, anadditive and an analgesic were made as follows. The opioid analgesicsufentanil citrate (obtained from National Institute on Drug Abuse), wasdesalted by adding ammonium hydroxide, extracted with n-hexane,collection of solvent and evaporation. The desalted sufentanil wasreconstituted in 20 μl of 90% ethanol containing approximately 4,500,000cpm of tritiated sufentanil (obtained from Jannsen Pharmaceutica,Belgium) to 2.0 mg/20 μl. The biodegradable protein SELP7 was dissolvedin deionized water to 42.3 mg (SELP7)/200 μl and spread into a thinlayer approximately 6 cm by 6 cm in area. Immediately thereafter, 22.5mg of finely pulverized powder of an additive, the fatty aciddimer:sabacic acid (FAD:SA in 1:4 ratio) was added to the center of theprotein solution area. Immediately thereafter, the sufentanil dissolvedin the ethanol was added very slowly to the mound of FAD:SA over aperiod of a few minutes, i.e., from about 1 minute to about 5 minutes.After the sufentanil solution had soaked into the FAD:SA powder, thecomponents were thoroughly mixed to form a coatable composition. Thecomposition was then coated onto a glass surface to form a film with athickness of approximately 0.1-0.2 mm. The film was allowed to dry atroom temperature until capable of forming a cohesive body, i.e., to asolvent content of from about 50% to 70%. The resultant cohesive bodywas rolled up and placed in a 3.5 mm diameter mold and compressed at8500 psi for 2 minutes to form a 3.5 mm diameter cylinder, approximately4 mm long and weighing 54.1 mg, utilizing the compression molding devicediscussed hereinabove. This device was then exposed to gamma irradiation(60-90 KRads) for sterilization and stored in a refrigerator (4° C.)until used within 8 weeks.

Example 7 Preparation of an Implantable Drug Delivery Device Comprising,Biodegradable Protein, an Additive and an Opioid Analgesic

Implantable drug delivery devices comprising a biodegradable protein, anadditive and an opioid analgesic were made as follows. The opioidanalgesic sufentanil citrate (obtained from National Institute on DrugAbuse) was desalted by adding ammonium hydroxide, extracted withn-hexane, collection of solvent and evaporation. The desalted sufentanilwas reconstituted in 20 μl of 90% ethanol containing approximately3,500,000 cpm of tritiated sufentanil (obtained from JannsenPharmaceutica, Belgium) to 2.0 mg/20 μl. The biodegradable protein SELP7was dissolved in deionized water to 15 mg (SELP7)/200 μl and spread intoa thin layer approximately 6 cm by 6 cm in area. Immediately thereafter,35.0 mg of finely pulverized powder of the additive glutamine, was addedto the center of the protein solution area. Immediately thereafter, thesufentanil dissolved in the ethanol was added very slowly to the moundof glutamine over a time period of a few minutes. After the sufentanilsolution had soaked into the glutamine powder, the components werethoroughly mixed to form a coatable composition. The composition wasthen coated onto a glass surface to form a film with a thickness ofapproximately 0.1-0.2 mm. The cast film was allowed to dry at roomtemperature until capable of forming a cohesive body, i.e., to a solventcontent of from about 50% to 70%. The resultant cohesive body was rolledup and placed in a 3.5 mm diameter mold and compressed at 8500 psi for 2minutes to form 3.5 mm diameter cylinders, approximately 2 mm long andweighing 39.1 mg, utilizing the compression molding device discussedhereinabove. This device was then exposed to gamma irradiation (60-90KRads) for sterilization and stored in a refrigerator (4° C.) until usedwithin 8 weeks.

Example 8 Preparation of an Implantable Drug Delivery Device Comprisinga Biodegradable Protein, an Additive and an Opioid Analgesic

Implantable drug delivery devices comprising a biodegradable protein, anadditive and an analgesic were made as follows. The opioid analgesicsufentanil citrate (obtained from National Institute on Drug Abuse), wasdesalted by adding ammonium hydroxide, extracted with n-hexane,collection of solvent and evaporation. The desalted sufentanil wasreconstituted in 20 μl of 90% ethanol containing approximately 4,500,000cpm of tritiated sufentanil (obtained from Jannsen Pharmaceutica,Belgium) to 2.0 mg/20 μl. The biodegradable protein SELP7 was dissolvedin deionized water to 42.3 mg (SELP7)/200 μl and spread into a thinlayer approximately 6 cm by 6 cm in area. Immediately thereafter, 22.5mg of finely pulverized powder of an additive, the fatty aciddimmer:sabacic acid (FAD:SA in 1:4 ratio) was added to the center of theprotein solution area. Immediately thereafter, the sufentanil dissolvedin the ethanol was added very slowly to the mound of FAD:SA over aperiod of a few minutes, i.e., from about 1 minute to about 5 minutes.After the sufentanil solution had soaked into the FAD:SA powder, thecomponents were thoroughly mixed to form a coatable composition. Thecomposition was then coated onto a glass surface to form a film with athickness of approximately 0.1-0.2 mm. The film was allowed to dry atroom temperature until capable of forming a cohesive body, i.e., to asolvent content of from about 50% to 70%. The resultant cohesive bodywas rolled up and placed in a 3.5 mm diameter mold and compressed at8500 psi for 2 minutes to form 3.5 mm diameter cylinders, approximately4 mm long and weighing 54.1 mg, utilizing the compression molding devicediscussed hereinabove. This device was then exposed to gamma irradiation(60-90 KRads) for sterilization and stored in a refrigerator (4° C.)until used within 8 weeks.

Example 9 In Vitro Experiment with a Drug Delivery Device Comprising aBiodegradable Protein and an Enzyme

A single cylinder piece, prepared as described above in Example 1, wasadded to a reaction chamber in a spectrophotometer containing xanthine,cytochrome C and other reactants according to previously describedsuperoxide dismutase protocol (Sigma Quality Control Test Procedure EC1.15.1.1 “Enzymatic Assay of Superoxide Dismutase”) enzyme activity ofthe enzyme xanthine oxidase in the piece was calculated at 0.0005 deltaabsorbance min (absorbance measured at 550 mm where no enzyme activityproduces 0.00000 change in absorbance). In comparison to a 0.01 unitsolution of xanthine oxidase, which produced 0.0250 deltaabsorbance/min, the activity of the xanthine oxidase in the pieceequaled 1% of the control solution in a time period of only 3 minutes.Thus, this result indicates that the diffusional barrier provided by thebiodegradable polymeric matrix of the drug delivery device allows theenzyme to remain active from within the drug delivery device.

Example 10 In Vitro Experiment with a Drug Delivery Device Comprising aBiodegradable Protein and an Enzyme

In this assay system, xanthine oxidase, xanthine, cytochrome C and otherreactants were added together to produce a delta absorbance of0.0250/min. (Sigma Quality Control Test Procedure EC 1.15.1.1 “EnzymaticAssay of Superoxide Dismutase”). SOD activity is measured as theinhibition of the rate of reduction of ferricytochrome C by superoxide,observed at 550 nm, as described by J. McCord, I. J. Biol. Chem., 244,6049 (1969). The addition of a SOD containing piece, produced asdescribed in Example 2 hereinabove, reduced the reaction to 0.0233 deltaabsorbance/min. Since 1 unit SOD will inhibit the reaction of cytochromeC by 50% in a coupled system using xanthine oxidase, it can bedetermined that the activity of the SOD pellet equaled 0.14 units ofSOD. This activity represents about 2% of the SOD loaded into thebiodegradable protein matrix of the drug delivery device. Thus, thisresult indicates that the diffusional barrier provided by thebiodegradable polymeric matrix of the drug delivery device allows theenzyme to remain active from within the drug delivery device.

Example 11 In Vivo Experiment with a Drug Delivery Device Comprising aBiodegradable Protein and Lipospheres

The drug delivery devices comprising a biodegradable protein andlipospheres produced according to Example 3 hereinabove were surgicallyimplanted next to the sciatic nerve of one young adult male SpragueDawley rat (200-250 g) as described previously by Masters in D. B.Masters et al., Anesthesiol., 79, 340 (1993). Briefly, the rat wasanesthetized with 50-75 mg/kg pentobarbital to allow faster recovery forbehavioral measurements. Bilateral posterolateral incisions were made inthe upper thighs and the sciatic nerves were visualized with care toavoid direct trauma. Drug delivery devices prepared as described inExample 3 were injected around the nerve on one leg, while no drugdelivery device was inserted in the contralateral leg to serve as acontrol. The fascia and muscle surrounding the administration site wasclosed over to partially restrict egress of the drug delivery device andthe entire wound area was lavaged with 0.5 cc of an antibiotic solution(5000 units/ml penicillin G sodium and 5000 μl/ml streptomycin sulfate).The experimenter performing subsequent thermal sensitivity testing andmotor capacity tests was unaware of which side received the drugdelivery device and which side received nothing.

After having the drug delivery device implanted, the rat was subjectedto periodic thermal sensitivity and motor capacity testing according tothe protocol described above. As shown in Table 1, the drug deliverydevices so implanted produced at least 4 days of local anesthetic block,i.e., a reduction in thermal sensitivity with a concurrent reduction inmotor capacity tests compared to the control leg.

TABLE 1 In vivo local anesthetic block produced by a drug deliverydevice comprising lipospheres (they themselves break down within thematrix) Time (hr) Thermal Sensitivity Tests Motor capacity (weightbearing) 0 100% ± 5% 100% ± 2% 2 427% 41% 4 560% 44% 20 196% 56% 26 216%62% 42 195% 79% 48 180% 77% 96 126% 75% 120 105% 76%

Example 12 In Vivo Experiment with a Drug Delivery Device Comprising aBiodegradable Protein and Two Pharmacologically Active Agents

Three “first drug delivery devices” prepared according to Example 4,i.e. comprising 6 mg of capsaicin and 6 mg dexamethasone were implantednext to the sciatic nerve of one young adult male Sprague Dawley ratusing the procedures described above in Example 7. The rat was monitoredfor a period of 624 hours. The results of this experiment are shown inTable 2, below. The first drug delivery devices produced strong thermalsensitivity, but no reduced motor capacity, for 6 days. Because the ratshowed some weight loss, the devices were removed on day 6. The ratcontinued to show a strong reduction in thermal sensitivity for the next14 days before returning to baseline response levels. In comparison tothe contralateral control leg, no reduced motor capacity was detected.Therefore, a very strong sensory neural blockade (analgesia) wasobtained by placement of these matrices without associated motordeficits.

TABLE 2 In vivo local anesthetic block produced by a drug deliverydevice incorporating 6 mg Capsaicin and 6 mg Dexamethasone ThermalSensitivity Test Time (hr) (experimental/control) Motor Capacity (weightbearing) −48 0.98 nd −24 0.98 0.99 −1 1.02 1.01 2 2.47 nd 4 2.04 0.97 241.80 0.95 48 2.72 1.01 96 1.94 0.86 144 2.86 0.91 168 2.34 0.97 192 2.190.99 216 3.04 1.00 264 2.59 1.00 288 1.76 1.05 312 1.58 0.99 318 2.550.99 336 2.06 1.01 360 1.65 0.98 384 1.65 0.99 432 2.16 0.99 456 1.351.01 480 0.92 0.99 504 1.10 1.01 528 0.98 1.02 552 1.38 1.00 624 1.071.01

Five “second drug delivery devices,” i.e., comprising 6 mg of capsaicinand 1.2 mg dexamethasone, prepared as described above in Example 4 wereimplanted next to the sciatic nerve of individual rats, where theyproduced a strong reduction in thermal sensitivity with no concurrentreduction in motor capacity for several days to weeks. All 5 rats showedsome weight loss, but far less than that observed with implantation ofthe first devices.

The results of this experiment are shown in Table 3, below. As shown, avery strong reduction in thermal sensitivity was obtained byimplantation of these devices without a concurrent reduction in motorcapacity. As is shown, all rats showed similar effects with variousdurations, i.e., no rats showed motor deficits. Lower doses of capsaicinand dexamethasone showed similar results.

TABLE 3 In vivo local anesthetic block produced by a drug deliverydevice incorporating 6 mg Capsaicin and 1.2 mg Dexamethasone Time (hr)Thermal Sensitivity Tests Motor capacity (weight bearing) −48 1.13 1.00−24 0.96 0.99 −1 1.02 1.02 2 2.72 1.02 4 3.77 1.00 24 2.50 1.17 48 2.861.00 96 2.72 0.96 120 1.78 1.01 144 3.05 1.01 168 2.06 0.98 192 1.821.00 216 1.74 1.03 288 3.14 1.00 312 2.88 1.00 336 2.17 1.01 360 1.830.99 456 1.33 480 1.22 0.99 504 1.85 1.01 528 1.72 0.99 552 1.92 1.01624 2.42 0.99 672 2.13 0.97 792 1.50 1.01 840 1.24 0.99 888 1.49 1.01984 1.36

Example 13 In Vitro Experiment with an Injectable Drug Delivery DeviceComprising a Biodegradable Protein, an Additive and an Opioid Analgesic

Four pellets, prepared as described in Example 5, were each added toseparate glass vials treated with a silicone coating (commerciallyavailable under the trade designation “Sigmacote” from Sigma ChemicalCompany, St. Louis, Mo.) to prevent loss of tritiated sufentanil. Thepellets were added to the glass vials filled with 15 ml of 0.1 Mphosphate buffered saline (pH 7.4), and then were incubated at 37° C.with agitation. At specific time intervals, 20 μl samples were taken intriplicate from each glass vial and measured for radioactive sufentanilusing a scintillation counter. As shown in FIG. 8, each of the fourmatrices produced at least 9 days of sufentanil release following afirst order release rate.

Example 14 In Vitro Experiment with an Implantable Drug Delivery DeviceComprising a Biodegradable Protein, an Additive and an Opioid Analgesic

A single pellet, prepared as described in Example 6 was added to a glassvial treated with a silicone coating (commercially available under thetrade designation “Sigmacote” from Sigma Chemical Company, St. Louis,Mo.) to prevent loss of tritiated sufentanil. The glass vial was filledwith 15 ml of 0.1 M phosphate buffered saline (pH 7.4), and incubated at37° C. with agitation. At specific time intervals, 20 μl samples weretaken in triplicate and measured for radioactive sufentanil using ascintillation counter. As shown in Table 4, this 3.5 mm diametercylinder matrix produced at least 75 days of sufentanil releasefollowing near zero-order release rate kinetics.

TABLE 4 In Vitro Release Study of Implantable Drug Delivery DeviceComprising a Biodegradable Protein, an Additive and an Opioid AnalgesicScintillation Time Counts Cumulative (hr) (cpm) Release (%)* 1 590501.48 4 26883 3.17 10 228667 5.72 28 263650 6.59 49 415150 10.38 73455000 11.38 120 561517 14.04 200 583333 14.58 251 619283 15.48 299653517 16.34 428 751517 18.79 603 901483 22.54 793 1281183 32.03 10301645650 41.14 1199 1810450 45.26 1368 2093083 52.33 1536 2532467 63.311704 3205867 80.15 1899 3446133 86.15 2003 3528650 88.22 2239 368971792.24 *Based on total expected counts = 4,500,000

Example 15 In Vitro Experiment with an Implantable Drug Delivery DeviceComprising a Biodegradable Protein, an Additive and an Opioid Analgesic

A single pellet, prepared as described in Example 7 was added to a glassvial treated with a silicone coating (to prevent loss of tritiatedsufentanil commercially available under the trade designation“Sigmacote,” from Sigma Chemical Company, St. Louis, Mo.). The glassvial was filled with 15 ml of 0.1 M phosphate buffered saline (pH 7.4),and incubated at 37° C. with agitation. At specific time intervals, 20μl samples were taken in triplicate and measured for radioactivesufentanil using a scintillation counter. As shown in Table 5, this 3.5mm diameter cylinder matrix produced approximately 2 days of sufentanilrelease. The addition of glutamine facilitated the release of sufentanilout of the matrix.

TABLE 5 In Vitro Release Study of Implantable Drug Delivery DeviceComprising a Biodegradable Protein, an Additive and an Opioid AnalgesicTime (hr) Scintillation Counts (cpm) Cumulative Release 1 59050 1.48 2671133 19.29 4 1495667 43.00 10 2230283 64.11 28 2908267 83.61 493346450 96.20 73 3422867 98.40 120 3439183 98.87 200 3430783 98.63leftover cpm in pellet 47792 *total cpm 3478575

Example 16 In Vivo Experiment with Drug Delivery Devices Comprising aBiodegradable Protein, an Additive and an Opioid Analgesic

The drug delivery devices comprising a protein (SELP7), an additive(FAD:SA), and an opioid analgesic (sufentanil), produced according toExample 5 hereinabove, were injected into the left side of the epiduralspace adjacent to spinal cord at the fifth lumbar vertebrae in 2 youngadult male Sprague Dawley rats. All rats underwent pre-testing forthermal sensitivity tests and motor capacity tests as describedhereinabove. The rats were anesthetized with halothane (4% induction, 2%maintenance) and prepared for spinal injection by creating a sterilesurgical field over the dorsal aspect of the lower lumbar vertebralcolumn. The placement of the drug delivery devices was in closeproximity to the left dorsal root ganglion and nerve root at lumbarlevel 5, which is associated with nerve input from the left hind paw viathe sciatic nerve. After needle insertion validation, drug deliverydevices were loaded into an 18 gauge Tuohy epidural needle forinjection, most commonly used by anesthesiologists for spinaladministration of drug solutions. Before injection of the implants intothe epidural space, validation of the space was carried out by x-raytechniques to locate the tip of the needle using an opaque catheter andsmall x-ray machine. Aspiration of the space occupied by the catheterwas also used to validate that it was in the dry epidural space and notthe subdural space which is filled with cerebrospinal fluid. The dosagedelivered from the drug delivery devices was adjusted by administeringmore than one implant into the epidural space. To test for a doseresponse effect, rat F043 received two drug delivery devices containingsufentanil and rat F045 received 6 drug delivery devices containingsufentanil. In this experiment a third rat, F046, was used as a controland received two control devices via the same epidural administrationtechnique. The control devices were made by the same coatablecomposition technique using the same quantities of biodegradable protein(SELP7), additive FAD:SA, deionized water and ethanol without thepresence of sufentanil. The results of this experiment are shown inTable 6, below, where time is in hours relative to epiduraladministration of the drug delivery devices. Rats F043 and F045 showedprolonged opioid analgesia for approximately 9-12 days in thermalsensitivity tests, performed as described hereinabove, i.e., increasedlatency (seconds) to remove their paws from a heated surface. Epiduralinjections of sufentanil citrate at highest possible doses withoutbecoming toxic (5-7 μg/kg), only produced 2 hours of measurable effectsto thermal sensitivity testing in three control rats.

TABLE 6 In Vivo Thermal Sensitivity Latency Tests for Drug DeliveryDevices Comprising a Protein, and a Polyanhydride Copolymer With andWithout an Opioid F043 (2 devices) F045 (6 devices) F046 (2 controldevices) Time (hr) Left Paw Right Paw Left Paw Right Paw Left Paw RightPaw −48 2 1.8 1.9 2 2.5 2.4 −24 2.4 2.1 2 2 2.7 2.3 −1 1.8 1.9 1.9 2.12.2 2.2 1 2.8 2.4 12 3.7 3.5 3.2 4 3 2 5.6 2.7 2.9 2.7 22 2.8 2.1 5.72.9 2.3 2.2 46 3.4 2.1 8.4 3 2.3 2.4 74 2.9 2.1 7.1 2.5 nd nd 119 2.81.8 6.8 2.4 2.5 2.4 144 nd nd 9.7 2 2.4 2.3 166 nd nd 7.5 2.2 2.5 2.4189 2.7 1.9 10.1 2.1 nd nd 211 3.1 2.1 5.6 2.5 nd nd 289 2.9 2.3 2.6 1.8nd nd 314 2.8 2 2.3 1.9 nd nd 337 2.5 1.8 1.9 1.9 nd nd 391 3 2.1 1.81.9 nd nd 435 2 2.1 1.8 1.7 nd nd 457 2.1 1.8 1.8 1.9 nd nd 482 nd nd 22 nd nd *nd = not determined; Testing was stopped after rat returned topre-device response level.

Example 17 Experiment with Drug Delivery Devices Comprising aBiodegradable Protein Matrix that Includes a Controlled ReleaseMechanism

Two types of drug delivery devices were prepared by compressing crystalsof Blue dextran or Gadolinium gadopentetate dimeglumine (Gd-DPTA)(Magnevist) in a polyanhydride copolymer of a 5:1 fatty acid dimer oferucic acid to sebacic acid and then coated by the same copolymer toproduce an insert. Following production of the insert, the insert wasencapsulated by compression molding in a protein matrix of collagen. Theblue dextran or Gd-DPTA & MRI was utilized to verify that the ultrasoundtriggered device was releasing its drug agents. Each drug deliverydevice had a diameter of 6 mm and a length of 7 mm. The drug deliverydevice including blue dextran was submerged in water and held in placewith monofilament. Once positioned in water, the drug deliver device wastriggered by a focused ultrasound pulse of 50 watts for 5 seconds andwas visually observed. FIG. 18 is a before and after depiction of thedrug delivery device that includes a release mechanism. The top panel ofFIG. 18 is an end and side view of the drug delivery device beforeultrasound triggering of the blue dextran polymer insert. The bottompanel is a view of the drug delivery device after ultrasound triggering.

FIGS. 19 and 20 depict the ultrasound triggering of a drug deliverydevice including a Gd-DPTA copolymer insert. FIG. 19 is an illustrationof two Gd-DPTA drug delivery devices contained in an agar gel positioned5 mm apart. The figure depicts the triggering of the targeted drugdelivery device with a focused ultrasound pulse of 50 watts for 5seconds. The Gd-DPTA was observed by Magnetic Resonance Imaging. TheGadolinium is shown to release from the drug delivery device in greateramounts over time.

FIG. 20 illustrates a time progression depiction of a drug deliverydevice including a Gd-DPTA copolymer insert that has been triggered by afocused ultrasound pulse of 50 watts for 5 seconds. The first frame at 0min is taken immediately before the ultrasound pulse. The followingframes sequentially illustrate the release progression of the Gd-DPTAinto the agar gel.

Example #18 Preparation of Collagen:Elastin (4:1 ratio) Tubular Grafts

In the preparation of the vascular tubes, Collagen:Elastin was used in a4:1 ratio and mixed with sterilized saline in amount equal to 600% theweight of the combined collagen and elastin (e.g., 80 mg collagen+20 mgelastin in 600 microliters of water). The material was mixed togetherand immediately thereafter, the pH was adjusted with drops of 0.1N and0.5N NaOH until pH indicator strips read 7.4 pH. The material was thenpartially dried at room temperature until it was to a state where it wascohesive unto itself and was then subsequently formed into a cohesivebody. The cohesive body was loaded into the mold were a mandrel insertwould receive the cohesive body as mechanically applied pressure forcedthe cohesive body over the mandrel with a final pressure equal to 5,000psi for a period of 10 minutes. The result was the formation of a tubearound the mandrel where the tube wall thickness was 0.2 mm and thelength of the tube was 1 cm. While the protein matrix tube was still onthe mandrel insert, it was submersed in 1% glutaraldyhde solution for 2minutes, resulting in partial cross linking of the outside of the tube.After 2 minutes, the tube-mandrel insert was submersed in saline for 1minute then it was subjected to a 15 minute submersion in a 0.1 Mphosphate buffered saline solution containing 1% glutamine and 1%glycine. The tube was then slipped off the mandrel, where the mandrelwas made with a slope of 0.001 inches over the 1 cm length to ease theremoval of the protein matrix tube. Also, before the mandril was placedin the mold it was coated with a slippery substance (e.g., glycol orTriton-X100). Finished tubes were stored in saline and sterilized with10-20 KRADS of gamma irradiation from a cesium source.

The following table includes vascular tubes with various compositionsprepared by following the procedure described above.

Composition Ratio Solvent pH Pressure Len./Dia.*/Wall Cross-Linking A)collagen:elastin (4:1) saline 7.4 15000 psi 1 cm/2 mm/0.2 mm outsidesurface B) collagen:elastin (4:1) saline 7.4 15000 psi 1 cm/2 mm/0.2 mmnone C) collagen:elastin (4:1) saline 7.4 15000 psi 1 cm/2.4 mm/0.2 mminside tube D) collagen:elastin (4:1) 9% NaCl 7.4 15000 psi 1 cm/2mm/0.2 mm outside surface E) collagen:elastin:heparin (4:1:1) saline 7.415000 psi 1 cm/2 mm/0.2 mm outside surface F) collagen:elastin (1:1)saline 7.4 15000 psi 1 cm/2 mm/0.2 mm outside surface G)heparin:elastin:collagen (1:4:15) saline 7.4 15000 psi 1 cm/2 mm/0.2 mmoutside surface H) elastin:albumin:collagen (4:1:1) saline 7.4 15000 psi1 cm/2 mm/0.2 mm outside surface I) collagen (1) saline 5 15000 psi 1cm/2 mm/0.2 mm outside surface J) chondroitin:elastin:collagen: (1:4:15)saline 7.4 15000 psi 1 cm/2 mm/0.2 mm outside surface K)collagen:albumin:elastin (2:2:1) saline 7.4 15000 psi 1 cm/2 mm/0.2 mmoutside surface L) collagen:albumin:elastin (2:1:2) saline 7.4 15000 psi1 cm/2.4 mm/0.2 mm inside surface M) collagen:albumin:elastin:glutamine(2:2:1) saline 5 15000 psi 1 cm/2 mm/0.2 mm outside surface N)elastin:albumin (1:3) saline 7.4 15000 psi 1 cm/2 mm/0.2 mm outsidesurface *diameter of interior of tube

Example #19 Endothelial Cell Seeding of Tubular Vessels

For this experiment, the protein tubes were produced by the methoddescribed in method #18. The endothelial used in the culture are humanumbilical vein endothelial cells (HUVEC). The tubes were seeded withthese cells in order to obtain a confluent endothelial monolayer withinthe lumen of the protein tubes. To obtain a high-density culture, thetubes were first cultured with these cells using standard culturingtechniques that are known in the discipline. The cells were cultured ona plastic dish that is two times lager than the surface area of theprotein tube's lumen. Next, the cells were detached from the culturedish using a trypsin/EDTA solution obtained from ICN Pharmaceuticals,Inc. The cells are then seeded into the lumen of the protein tube. Fourhours after seeding, the nonattached cells were be removed. Tubes werethen incubated at 37° C. under 5% CO2 and 95% air atmosphere in astandard solution of DMEM. The medium was replaced at least every otherday for 4-7 days. Cells have been found to adhere and grow to aconfluent monolayer on tubes made of collagen and elastin (4:1 ratio),100% collagen, and heparin:elastin:collagen (1:4:15).

Example #20 Preparation of Wound Healing Device (Tissue Graft; Wafer)

Dried bovine type I collagen (ICN Biomedicals, Aurora, Ohio) wassolubilized using vitrogen and distilled water added in a dropwisemanner. Vitrogen was continually added to ensure that the collagen didnot dry out before all of the collagen had solubilized. Once thecollagen had dissolved, the mixture was allowed to dry until it attaineda cohesive state. The collagen was then rolled into a cylinder andplaced in a brass mold between two stainless steel inserts. The collagencylinder was then compressed at 5700 psi for 10 minutes using apneumatic press. The cylinder was removed and divided into wafers usinga razor blade. Wafers were approximately 0.5 mm thick and were 6 mm indiameter unless otherwise stated. Wafers were then recompressed usingthe pneumatic press for 10 minutes at either 5740 or 28700 psi(henceforth referred to as low and high pressure, respectively). Somewafers were then removed from the brass mold and stored at 4° C. untilthey were crosslinked. After crosslinking and prior to use in cellculture experiments, all wafers were sterilized using a Cesiumirradiator. FIG. 21 is a magnified view of a noncrosslinked wafer afterit has been incubating overnight in phosphate buffered saline. FIG. 22is a magnified view of a crosslinked wafer after it has been incubatingovernight in phosphate buffered saline.

Example #21 Glutaraldehyde Crosslinking Wound Healing Device (TissueGraft; Wafer)

A 1% glutaraldehyde solution (Sigma, St. Louis, Mo.) was used forcrosslinking wafers. A single wafer was incubated for 1, 3, 5, 15, or 30minutes in 1 ml of 1% glutaraldehyde solution in 1×PBS. Samples werethen washed in 1 ml of 1 PBS for 10 minutes. This washing procedure wasrepeated two more times. A revised washing protocol was developed inlight of evidence that the cells were dying due to cytotoxic effects ofglutaraldehyde. In this new process, glutaraldehyde was removed from thesamples and then wafers were transferred to a clean plastic tube. Theywere then washed in 5 ml of 1×PBS for 4 hours. The PBS was removed and 5ml of fresh 1×PBS was added for a second washing for 8 hours(overnight). The PBS was again removed and the wafers were washed for 2hours prior to cell seeding in a modified 1×PBS solution, whichconsisted of 1 mM glycine, and 1:100 dilution of vitrogen. This lastwash was intended to bind up any residual glutaraldehyde and therebyeliminate the cytotoxic effects of free glutaraldehyde. Collagen wafersthat did no undergo crosslinking were washed in the same buffers andused as controls.

Example #22 Mechanical Testing System (MTS) of Protein Matrix MaterialMTS Testing:

Six wafers from each experimental group were tested to determinestructural and mechanical properties. Sample thickness was measuredusing a Fowler micrometer (accurate to 0.1 mm). Cross-sectional areaswere calculated by assuming a rectangular cross-section. The UTS andmodulus (slope of the stress-strain curve) were determined from thestress-strain curves of the collagen wafers. Stress was calculated bydividing the force by initial cross-sectional area. Stress-strain curvesfor wafers were determined an MTS Microbionix biomechanical testercontrolled by TestStar/TestWare software. Wafers were tested using agauge length of 0.5 mm and a strain rate of 0.8 mm/s after rehydrationfor 10 minutes in phosphate buffered saline. The instrument was operatedin a dynamic mode at room temperature. The wafers were removed from thesolution immediately before testing and mounted onto the screw clamps. Awafer was mounted using two parallel screw clamps such that each clampsecured a segment of the wafer with a gauge length of 0.5 mm. The clampswere connected to the actuator and a 5-Newton force transducer of theMTS Microbionix testing system allowing continuous measurement of thestress response to a constant strain rate in the radial direction inextension by separating the screw clamps at a constant speed. Stress wascalculated by dividing the force generated during extension by theinitial wafer cross-sectional area (approximated by multiplying waferthickness by the wafer diameter). Strain was calculated as the naturallog of the ratio of the extended distance over the gauge length. TheYoung's modulus was determined by measuring the slope of thestress/strain curve between strains of 0.2 and 0.8. Ultimate TensileStrength (UTS) represents that largest stress value sustained by thewafer during testing.

Crosslinking and Pressure Effects on the Mechanical Properties ofCollagen Devices:

Young's modulus and UTS were assessed used to characterize themechanical properties of the collagen DDS. An increase in Young'smodulus was seen as the duration of glutaraldehyde crosslinkingincreased for both low (5700 psi) and high (28,700 psi) psi compressiveloads (FIG. 23). For the low psi wafers the increase was significantbetween 0 and 3, 0 and 15, and 0 and 30, 1 and 15, 1 and 30, 3 and 30, 5and 30 minutes, and 15 and 30 minutes based on ANOVA analysis. For thehigh psi wafers, the increase was significant between all paired timepoints except 1 and 3, 1 and 5, and 3 and 5 minutes. In addition, therewas no significant difference between the high and low psi systems atany of the crosslinking times based on ANOVA analysis.

The UTS of the collagen systems also increased as the length ofcrosslinking time increased for both the low psi and high psi loadlevels (FIG. 24). For the low psi wafers this difference was significantbetween all pairs of time points except 1 and 3, 1 and 5, 1 and 15, 3and 5, and 3 and 15 minutes. For the high psi wafers, the increase wassignificant between all pairs of time points except 1 and 3 minutesbased on ANOVA analysis. There was no significant difference between thelow psi and high psi system at any of the crosslinking times.

Example #23 Dissolution of Collagen Protein from Collagen Wafers

FIGS. 25-28 depict the results of tests performed regarding dissolutionof collagen from collagen wafers made with medium (12,000 psi), high(20,000 psi) and high (28,000 psi) pressures in a compression chamberand with various amounts of crosslinking. The wafers were crosslinkedwith 1% glutaraldehyde for 0, 1, 10, and 30 minutes corresponding toFIGS. 25-28, respectively. The collagen wafers were analyzed by placingthem in phosphate buffered saline in a 15 ml conical Falcon tube (pH7.4, 37° C.). The Falcon tube was then place in a shaking incubator at37° C. and set to slow agitation. At various time points samples of thesolution were tested by BCA protein assay (Pierce Company) for proteincontent and recorded.

Example #24 Mechanical Testing System (MTS) of Protein Matrix Material(Vascular Tubes) MTS Testing:

A vascular tube was tested to determine structural and mechanicalproperties. Sample thickness was measured using a Fowler micrometer(accurate to 0.1 mm). Stress-strain curves for tubes were determined anMTS Microbionix biomechanical tester controlled by TestStar/TestWaresoftware. The tube was wet with a phosphate buffered saline. Theinstrument was operated in a dynamic mode at room temperature. The tubewas mounted onto prongs made to fit the inside diameter of the tube. Theprongs were mounted to the actuator and a 5-Newton force transducer ofthe MTS Microbionix testing system allowing continuous measurement ofthe stress response to a constant strain rate in the radial direction inextension by separating the prongs at a constant speed. Stress wascalculated by dividing the force generated during spreading of the tubewalls (approximated by multiplying wall thickness by the tube walldiameter). Strain was calculated as the natural log of the ratio of theextended distance over the gauge length. Ultimate Tensile Strength (UTS)represents that largest stress value sustained by the wafer duringtesting. The UTS that resulted was equal to 192.6 mmHg.

Example #25 Mechanical and Hydraulic Testing System (MTS) of ProteinMatrix Material (Tubular Grafts)

A vascular tube was prepared as described above using a mixture ofcollagen:albumin:elastin (ratio 2:2:1) (pH 7.4; 2 mm inner diameter).FIG. 29 depicts an embodiment of the vascular tube. The tube was placedover polyethylene hose, tied with silk suture material and cemented withadhesive. The tube was then visually tested for durability andcompliance by twisting. FIG. 30 depicts the vascular tube tested fordurability and compliance. FIG. 31 depicts both sides of a vascular tubetested for hydraulic pressure. The polyethylene hose was attached to aTygon S-50-HL class V1 hose that was attached to a peristaltic pump thatcirculated phosphate buffered saline (PBS) through the hose and tube at3.5 ml/min. It was found that back pressure of over 200 mm Hg could begenerated several times without damaging the vascular tube. FIG. 32depicts, at the arrows, the vascular tube bulging in response to over200 mm Hg back pressure. FIG. 32 also illustrates that the back pressurecould have been greater, but for leakage occurring at the vascular tubeand polyethylene hose junction. In similar replicate tubes, it was foundthat no leaking occurred after 72 hours of constant circulation of PBSfluid.

Example #26 Preparation of Poly(vinyl-alcohol) (PVA) Particles inProtein Matrix Wafers

In this study PVA super hydrolyzed (99.3% M.W. 106,000-110,000,viscosity of 4% aqueous solution 55-65 cps at 20° C.) and recombinanthuman epidermal growth factor (hEGF) (R&D System) were used. A 4%solution of PVA (J.T. Baker) in distilled water was dissolved 1 hour at85° C. and added to a hEGF solution, which was dissolved into thedistilled water (50 μg/ml) and dried at 40° C. at vacuum oven overnight. The film was pulverized and then sieved to separate EGF-PVAparticles into various groups by size. The size of final particles was250-500 μm in diameter.

Formulation of Protein Matrix Containing Collagen:

Collagen (80 mg) (Type I, calf skin) (ICN Biomedicals Inc.) wasdissolved in 700 μl vitrogen and 200 μl distilled water and spread anddried entirely on glass plate until spread protein became cohesive. Oncecohesive body was formed the EGF-PVA particles (6 mg) were added to thecohesive body and rolled into a cylinder and made into a protein matrixwafer form by compression molding at 2000 psi. The wafers werecross-linked for 0, 3, 15, 30, or 60 minute in a 1% glutaraldehydesolution and subsequently rinsed 3 times in a 5 ml buffer solution (PBS)for three minute each time. Then EGF-PVA particles and cross-linked andnon-cross-linked matrices were sterilized 30 minute by Cesium-137irradiation (>10K RADS).

Release Study:

EGF-PVA particles, cross-linked wafers and non-cross-linked wafers wereincubated on the thermal rocker at 37° C. in 1 ml of PBS or William's Emedium solution. One ml samples were collected and replaced with freshmedium solution from each tube at 1, 4, 8, 24, 48, 72, 96, 120, 144,192, 240 hour time intervals. The EGF release was monitored in vitrousing a specific enzyme linked immunosorbent assay (ELISA) for bothparticle and matrices.

ELISA Assay:

The release of hEGF was measured using ELISA. The cytokine antibodypairs were used for construction of ELISAs. The captured antibody wasmonoclonal anti-human EGF antibody (MAB 636) (500 μg) (R&D System) anddetection antibody was biotinylated anti-human EGF antibody (BAF236) (50μg) (R&D System). The wells of a 96-well titertek plate (Polysorb, NuncPlasticware) were coated with monoclonal anti-human EGF antibody in PBSsolution. Sample or standards were added in an appropriate diluent perwell. The biotinylated detection antibody was diluted in the appropriatediluent (0.1% BSA, 0.05% Tween 20 in Tris-buffered Saline pH 7.3 (20 mMTrizma base, 150 mM NaCl), and added to each well. The plate was coveredwith an adhesive strip and incubated 2 hours at room temperature.Streptavidin HRP(Zymed) 1/2500 of a 1.25 mg/ml solution or equivalent)was then added to each well followed by a substrate solution (H₂O₂) anddeveloper ABTS (2,2′Azino-di[3-ethylbenzthiazoline-6-sulfonate])(Boehringer-Mannheim). The assay was incubated for 20-30 minutes at 37°C. The optical density was determined for each well in the plate within30 minutes, using microplate reader 450 nm. FIG. 33 depicts the resultsof the hEGF release study from the PVA particles used in the proteinmatrix wafers. The results of this study show that crosslinking theprotein matrix decreases the release of hEGF. It was also determinedfrom subsequent studies that the hEGF released has biological activityin in-vitro cell culture studies using hepatocytes.

While the invention has been described in conjunction with specificembodiments thereof, it is evident that many alternatives,modifications, and variations will be apparent to those skilled in theart in light of the foregoing description. Accordingly, it is intendedto embrace all such alternatives, modifications, and variations, whichfall within the spirit and broad scope of the invention.

1. A tissue graft comprising one or more biocompatible proteinmaterials, combined with one or more biocompatible solvents, wherein theprotein material(s) and biocompatible solvent(s) are formed into acohesive body having a solvent content of about 20% to 80% prior tocompression and the cohesive body is compressed at a pressure of about100 psi to 100,000 psi to remove bulk biocompatible solvent and generateadditional intermolecular and intramolecular forces between one or moreof the protein material(s) and solvent(s) to form the tissue grafthaving a solvent content of about 10% to 60%.
 2. The tissue graft ofclaim 1 wherein the biocompatible proteins may be natural, synthetic orgenetically engineered.
 3. The tissue graft of claim 2 wherein thebiocompatible proteins are natural proteins selected from the groupconsisting of elastin, collagen, albumin, keratin, fibronectin, silk,silk fibroin, actin, myosin, fibrinogen, thrombin, aprotinin andantithrombin III.
 4. The tissue graft of claim 2 wherein thebiocompatible proteins are genetically engineered proteins made ofblocks selected from the group consisting of elastinlike blocks,silklike blocks, collagenlike blocks, lamininlike blocks,fibronectinlike blocks and silklike and elastinlike blocks.
 5. Thetissue graft of claim 1 wherein the one or more biocompatible solventsare selected from the group consisting of water, dimethyl sulfoxide(DMSO), biocompatible alcohols, biocompatible acids, oils andbiocompatible glycols.
 6. The tissue graft of claim 1 further comprisingone or more pharmacologically active agents, cells or a combinationthereof.
 7. The tissue graft of claim 6 wherein the one or morepharmacologically active agents are selected from the group consistingof analgesics, anesthetics, antipsychotic agents, steroids,antisteroids, corticosteroids, antiglacoma agents, antialcohol agents,anti-coagulants agents, genetic material, antithrombolytic agents,anticancer agents, anti-Parkinson agents, antiepileptic agents,anti-inflammatory agents, anticonception agents, enzymes agents, cells,growth factors, antiviral agents, antibacterial agents, antifungalagents, hypoglycemic agents, antihistamine agents, chemoattractants,neutraceuticals, antiobesity, smoking cessation agents, obstetric agentsand antiasmatic agents.
 8. The tissue graft of claim 1 furthercomprising one or more biocompatible polymeric materials.
 9. The tissuegraft of claim 8 wherein the one or more biocompatible polymericmaterials are selected from the group consisting of epoxies, polyesters,acrylics, nylons, silicones, polyanhydride, polyurethane, polycarbonate,poly(tetrafluoroethylene), polycaprolactone, polyethylene oxide,polyethylene glycol, poly(vinyl chloride), polylactic acid, polyglycolicacid, polypropylene oxide, poly(alkylene)glycol, polyoxyethylene,sebacic acid, polyvinyl alcohol, 2-hydroxyethyl methacrylate, polymethylmethacrylate, 1,3-bis(carboxyphenoxy)propane, lipids,phosphatidylcholine, triglycerides, polyhydroxybutyrate,polyhydroxyvalerate, poly(ethylene oxide), poly ortho esters, poly(amino acids), polycynoacrylates, polyphophazenes, polysulfone,polyamine, poly (amido amines), fibrin, graphite, flexiblefluoropolymer, isobutyl-based, isopropyl styrene, vinyl pyrrolidone,cellulose acetate dibutyrate, silicone rubber, and copolymers of these.10. The tissue graft of claim 1 wherein the tissue graft is crosslinkedwith one or more crosslinking agents.
 11. The tissue graft of claim 10wherein the tissue graft is crosslinked with one or more crosslinkingagents.
 12. The tissue graft of claim 1 wherein the tissue graft isselected from the group consisting of vessels, tubular grafts, trachealtubes, bronchial tubes, catheter functioning tubes, lung grafts,gastrointestinal segments; clear matrix grafts; heart valves; cartilage;tendons; ligaments, skin grafts, particles and pancreatic implantdevices.
 13. The tissue graft of claim 1 wherein the biocompatibleprotein materials are collagen and elastin.
 14. The tissue graft ofclaim 1 wherein the protein matrix material includes one or moreadditives in an amount up to 300% based upon the weight of thebiocompatible protein material.
 15. The tissue graft of claim 6 whereinthe one or more pharmacologically active agents are included in theamount of 0.001% to 200% based upon the weight of the biocompatibleprotein material.
 16. A method of replacing damaged, diseased or missingtissue comprising: providing a tissue graft comprising one or morebiocompatible protein materials, combined with one or more biocompatiblesolvents and one or more pharmacologically active agents, cells orcombinations thereof, wherein the protein material(s), biocompatiblesolvent(s) and pharmacologically active agent(s), cells or combinationsthereof are formed into a cohesive body having a solvent content ofabout 20% to 80% prior to compression and the cohesive body iscompressed at a pressure of about 100 psi to 100,000 psi to remove bulkbiocompatible solvent and generate additional intermolecular andintramolecular forces between one or more of the protein material(s),solvent(s) and/or active agent(s) to form the tissue graft having asolvent content of about 10% to 60%; and administering the tissue graftto a part of a patient's body that is damaged, diseased or missing. 17.The method of replacing damaged, diseased or missing tissue of claim 16wherein the biocompatible solvent is selected from the group consistingof water, dimethyl sulfoxide (DMSO), biocompatible alcohols,biocompatible acids, oils and biocompatible glycols.
 18. The method ofreplacing damaged, diseased or missing tissue of claim 16 furthercomprising one or more pharmacologically active agents, cells or acombination thereof.
 19. The method of replacing damaged, diseased ormissing tissue of claim 18 wherein the one or more pharmacologicallyactive agents are selected from the group consisting of analgesics,anesthetics, antipsychotic agents, steroids, antisteroids,corticosteroids, antiglacoma agents, antialcohol agents, anti-coagulantsagents, genetic material, antithrombolytic agents, anticancer agents,anti-Parkinson agents, antiepileptic agents, anti-inflammatory agents,anticonception agents, enzymes agents, growth factors, antiviral agents,antibacterial agents, antifungal agents, hypoglycemic agents,antihistamine agents, chemoattractants, neutraceuticals, antiobesity,smoking cessation agents, obstetric agents, antimicrobial agents,glycosaminoglycans, cancer agents, and antiasmatic agents.
 20. Themethod of replacing damaged, diseased or missing tissue of claim 18,wherein the pharmacologically active agent comprises a second,migration-vulnerable drug delivery device.
 21. The method of replacingdamaged, diseased or missing tissue of claim 16 further comprising oneor more biocompatible polymeric materials.
 22. The method of replacingdamaged, diseased or missing tissue of claim 20 wherein the one or morebiocompatible polymeric materials are selected from the group consistingof epoxies, polyesters, acrylics, nylons, silicones, polyanhydride,polyurethane, polycarbonate, poly(tetrafluoroethylene),polycaprolactone, polyethylene oxide, polyethylene glycol, poly(vinylchloride), polylactic acid, polyglycolic acid, polypropylene oxide,poly(alkylene)glycol, polyoxyethylene, sebacic acid polymers, polyvinylalcohol, 2 hydroxyethyl methacrylate polymers, polymethyl methacrylate,1,3 bis(carboxyphenoxy)propane polymers, lipids, phosphatidylcholine,triglycerides, polyhydroxybutyrate, polyhydroxyvalerate, poly(ethyleneoxide), poly ortho esters, poly (amino acids), polycyanoacrylates,polyphophazenes, polysulfone, polyamine, poly (amido amines), fibrin,graphite, flexible fluoropolymer, isobutyl based polymers, isopropylstyrene polymers, vinyl pyrrolidone polymers, cellulose acetatedibutyrate polymers, silicone rubber, and copolymers and combinations ofthese.
 23. The method of replacing damaged, diseased or missing tissueof claim 16 wherein all or a portion of the tissue graft is crosslinkedwith one or more crosslinking agents.
 24. The method of replacingdamaged, diseased or missing tissue of claim 23 wherein the one or morecrosslinking reagents are selected from the group consisting ofglutaraldehyde, p-Azidobenzolyl Hydazide, N-5-Azido2-nitrobenzoyloxysuccinimide, N-Succinimidyl6-[4′azido-2′nitro-phenylamino]hexanoate and 4-[p-Azidosalicylamido]butylamine.
 25. The method of replacing damaged, diseased or missingtissue of claim 16 wherein the tissue graft is selected from the groupconsisting of vessels, tubular grafts, tracheal tubes, bronchial tubes,catheter functioning tubes, lung grafts, gastrointestinal segments;clear matrix grafts; heart valves; cartilage; tendons; ligaments, skingrafts, particles, bone inserts, meshes, strips, sutures, dental plugs,tissue plugs, vertebrae inserts, vertebral discs, joints, bronchialtissue inserts, abdominal inserts, vascular inserts, port seals andpancreatic implant devices.
 26. The method of replacing damaged,diseased or missing tissue of claim 16 wherein the biocompatible proteinmaterials are collagen and elastin.
 27. The method of replacing damaged,diseased or missing tissue of claim 16 wherein the protein matrixmaterial includes one or more additives in an amount up to 300% basedupon the weight of the biocompatible protein material.
 28. The method ofreplacing damaged, diseased or missing tissue of claim 18 wherein theone or more pharmacologically active agents are included in the amountof 0.001% to 200% based upon the weight of the biocompatible proteinmaterial.
 29. A method of making a tissue graft, comprising the stepsof: (a) preparing a coatable composition comprising one or morebiocompatible protein materials and one or more biocompatible solvents;(b) forming the coatable composition into a cohesive body having asolvent content of about 20% to 80% prior to compression; and (c)compressing the cohesive body at a pressure of about 100 psi to 100,000psi to remove bulk biocompatible solvent to form a tissue graft having asolvent content of about 10% to 60%.
 30. The method of making a tissuegraft device of claim 29 wherein the biocompatible proteins are selectedfrom the group consisting of elastin, collagen, albumin, keratin,fibronectin, silk, silk fibroin, actin, myosin, fibrinogen, thrombin,aprotinin, antithrombin III, elastinlike blocks, silklike blocks,collagenlike blocks, lamininlike blocks, fibronectinlike blocks andsilklike, elastinlike blocks, collagen-heparin and collagen-chondroiten.31. The method of making a tissue graft of claim 29 wherein thebiocompatible solvent is selected from the group consisting of water,dimethyl sulfoxide (DMSO), biocompatible alcohols, biocompatible acids,oils and biocompatible glycols.
 32. The method of making a tissue graftof claim 29, wherein the tissue graft further includes one or morepharmacologically active agents, cells or a combination thereof.
 33. Themethod of making a tissue graft of claim 32 wherein the one or morepharmacologically active agents are selected from the group consistingof analgesics, anesthetics, antipsychotic agents, steroids,antisteroids, corticosteroids, antiglacoma agents, antialcohol agents,anti-coagulants agents, genetic material, antithrombolytic agents,anticancer agents, anti Parkinson agents, antiepileptic agents,anti-inflammatory agents, anticonception agents, enzymes agents, growthfactors, antiviral agents, antibacterial agents, antifungal agents,hypoglycemic agents, antihistamine agents, chemoattractants,neutraceuticals, antiobesity, smoking cessation agents, obstetricagents, antimicrobial agents, glycosaminoglycans, cancer agents, andantiasmatic agents.
 34. The method of making a tissue graft of claim 32,wherein the pharmacologically active agent comprises a second,migration-vulnerable drug delivery device.
 35. The method of making atissue graft of claim 29 further comprising one or more biocompatiblepolymeric materials.
 36. The method of making a tissue graft of claim 35wherein the one or more biocompatible polymeric materials are selectedfrom the group consisting of epoxies, polyesters, acrylics, nylons,silicones, polyanhydride, polyurethane, polycarbonate,poly(tetrafluoroethylene), polycaprolactone, polyethylene oxide,polyethylene glycol, poly(vinyl chloride), polylactic acid, polyglycolicacid, polypropylene oxide, poly(alkylene)glycol, polyoxyethylene,sebacic acid polymers, polyvinyl alcohol, 2 hydroxyethyl methacrylatepolymers, polymethyl methacrylate, 1,3 bis(carboxyphenoxy)propanepolymers, lipids, phosphatidylcholine, triglycerides,polyhydroxybutyrate, polyhydroxyvalerate, poly(ethylene oxide), polyortho esters, poly (amino acids), polycyanoacrylates, polyphophazenes,polysulfone, polyamine, poly (amido amines), fibrin, graphite, flexiblefluoropolymer, isobutyl based polymers, isopropyl styrene polymers,vinyl pyrrolidone polymers, cellulose acetate dibutyrate polymers,silicone rubber, and copolymers and combinations of these.
 37. Themethod of making a tissue graft of claim 29 further comprising the stepof crosslinking all or a portion of the tissue graft with one or moresuitable crosslinking agents.
 38. The method of making a tissue graft ofclaim 37 wherein the one or more crosslinking reagents are selected fromthe group consisting of glutaraldehyde, p-Azidobenzolyl Hydazide,N-5-Azido 2-nitrobenzoyloxysuccinimide, N-Succinimidyl6-[4′azido-2′nitro-phenylamino]hexanoate and 4-[p-Azidosalicylamido]butylamine.
 39. The method of making a tissue graft of claim 29 furthercomprising the step of processing the tissue graft into particles. 40.The method of making a tissue graft of claim 29 further comprising thestep of adjoining two or more tissue grafts to form a multi-layer tissuegraft.
 41. The method of making a tissue graft of claim 29 wherein thebiocompatible protein materials are collagen and elastin.
 42. The methodof making a tissue graft of claim 29 wherein the protein matrix materialincludes one or more additives in an amount up to 300% based upon theweight of the biocompatible protein material.
 43. The method of making atissue graft of claim 32 wherein the one or more pharmacologicallyactive agents are included in the amount of 0.001% to 200% based uponthe weight of the biocompatible protein material.